A biomimetic, peptide-mediated approach to inorganic nanostructure formation is of great interest as an alternative to industrial production methods. To investigate the role of peptide structure on silica (SiO2) and titania (TiO2) morphologies, we use the R5 peptide domain derived from the silaffin protein to produce uniform SiO2 and TiO2 nanostructures from the precursor silicic acid and titanium bis(ammonium lactato)dihydroxide, respectively. The resulting biosilica and biotitania nanostructures are characterized using scanning electron microscopy. To investigate the process of R5-mediated SiO2 and TiO2 formation, we carry out 1D and 2D solid-state NMR (ssNMR) studies on R5 samples with uniformly 13C- and 15N-labeled residues to determine the backbone and side-chain chemical shifts. 13C chemical shift data are in turn used to determine peptide backbone torsion angles and secondary structure for the R5 peptide neat, in silica, and in titania. We are thus able to assess the impact of the different mineral environments on peptide structure, and we can further elucidate from 13C chemical shifts change the degree to which various side chains are in close proximity to the mineral phases. These comparisons add to the understanding of the role of R5 and its structure in both SiO2 and TiO2 formation.

In nature, organisms including diatoms, radiolaria, and marine sponges use proteins, long chain polyamines, and other organic molecules to regulate the assembly of complex silica-based structures. Here, the authors investigate structural features of small peptides, designed to mimic the silicifying activities of larger proteins found in natural systems. LKα14 (Ac-LKKLLKLLKKLLKL-C), an amphiphilic lysine/leucine repeat peptide with an α-helical secondary structure at polar/apolar interfaces, coprecipitates with silica to form nanospheres. Previous 13C magic angle spinning studies suggest that the tetrameric peptide bundles that LKα14 is known to form in solution may persist in the silica-complexed form, and may also function as catalysts and templates for silica formation. To further investigate LKα14 aggregation in silica, deuterium solid-state nuclear magnetic resonance (2H ssNMR) was used to establish how leucine side-chain dynamics differ in solid LKα14 peptides isolated from aqueous solution, from phosphate-buffered solution, and in the silica-precipitated states. Modeling the 2H ssNMR line shapes probed the mechanisms of peptide preaggregation and silica coprecipitation. The resulting NMR data indicates that the peptide bundles in silica preserve the hydrophobic interior that they display in the hydrated solid state. However, NMR data also indicate free motion of the leucine residues in silica, a condition that may result from structural deformation of the aggregates arising from interactions between the surface lysine side chains and the surrounding silica matrix.

Intrinsic motions may allow HIV-1 transactivation response (TAR) RNA to change its conformation to form a functional complex with the Tat protein, which is essential for viral replication. Understanding the dynamic properties of TAR necessitates determining motion on the intermediate nanosecond-to-microsecond time scale. To this end, we performed solid-state deuterium NMR line-shape and T1Z relaxation-time experiments to measure intermediate motions for two uridine residues, U40 and U42, within the lower helix of TAR. We infer global motions at rates of ∼105 s–1 in the lower helix, which are much slower than those in the upper helix (∼106 s–1), indicating that the two helical domains reorient independently of one another in the solid-state sample. These results contribute to the aim of fully describing the properties of functional motions in TAR RNA.

Silaffins, long chain polyamines, and other biomolecules found in diatoms are involved in the assembly of a large number of silica nanostructures under mild, ambient conditions. Nanofabrication researchers have sought to mimic the diatom’s biosilica production capabilities by engineering proteins to resemble aspects of naturally occurring biomolecules. Such mimics can produce monodisperse biosilica nanospheres, but in vitro production of the variety of intricate biosilica nanostructures that compose the diatom frustule is not yet possible. In this study we demonstrate how LK peptides, composed solely of lysine (K) and leucine (L) amino acids arranged with varying hydrophobic periodicities, initiate the formation of different biosilica nanostructures in vitro. When L and K residues are arranged with a periodicity of 3.5 the α-helical form of the LK peptide produces monodisperse biosilica nanospheres. However, when the LK periodicity is changed to 3.0, corresponding to a 310 helix, the morphology of the nanoparticles changes to elongated rod-like structures. β-strand LK peptides with a periodicity of 2.0 induce wire-like silica morphologies. This study illustrates how the morphology of biosilica can be changed simply by varying the periodicity of polar and nonpolar amino acids.

Extracellular matrix proteins adsorbed onto mineral surfaces exist in a unique environment where the structure and dynamics of the protein can be altered profoundly. To further elucidate how the mineral surface impacts molecular properties, we perform a comparative study of the dynamics of nonpolar side chains within the mineral-recognition domain of the biomineralization protein salivary statherin adsorbed onto its native hydroxyapatite (HAP) mineral surface versus the dynamics displayed by the native protein in the hydrated solid state. Specifically, the dynamics of phenylalanine side chains (viz., F7 and F14) located in the surface-adsorbed 15-amino acid HAP-recognition fragment (SN15: DpSpSEEKFLRRIGRFG) are studied using deuterium magic angle spinning (2H MAS) line shape and spin–lattice relaxation measurements. 2H NMR MAS spectra and T1 relaxation times obtained from the deuterated phenylalanine side chains in free and HAP-adsorbed SN15 are fitted to models where the side chains are assumed to exchange between rotameric states and where the exchange rates and a priori rotameric state populations are varied iteratively. In condensed proteins, phenylalanine side-chain dynamics are dominated by 180° flips of the phenyl ring, i.e., the “π flip”. However, for both F7 and F14, the number of exchanging side-chain rotameric states increases in the HAP-bound complex relative to the unbound solid sample, indicating that increased dynamic freedom accompanies introduction of the protein into the biofilm state. The observed rotameric exchange dynamics in the HAP-bound complex are on the order of 5–6 × 106 s–1, as determined from the deuterium MAS line shapes. The dynamics in the HAP-bound complex are also shown to have some solution-like behavioral characteristics, with some interesting deviations from rotameric library statistics.

The use of biomimetic approaches in the production of inorganic nanostructures is of great interest to the scientific and industrial community due to the relatively moderate physical conditions needed. In this vein, taking cues from silaffin proteins used by unicellular diatoms, several studies have identified peptide candidates for the production of silica nanostructures. In the current article, we study intensively one such silica-precipitating peptide, LKα14 (Ac-LKKLLKLLKKLLKL-c), an amphiphilic lysine/leucine repeat peptide that self-organizes into an α-helical secondary structure under appropriate concentration and buffer conditions. The suggested mechanism of precipitation is that the sequestration of hydrophilic lysines on one side of this helix allows interaction with the negatively charged surface of silica nanoparticles, which in turn can aggregate further into larger structures. To investigate the process, we carry out 1D and 2D solid-state NMR (ssNMR) studies on samples with one or two uniformly 13C- and 15N-labeled residues to determine the backbone and side-chain chemical shifts. We also further study the dynamics of two leucine residues in the sequence through 13C spin–lattice relaxation times (T1) to determine the impact of silica coprecipitation on their mobility. Our results confirm the α-helical secondary structure in both the neat and silica-complexed states of the peptide, and the patterns of chemical shift and relaxation time changes between the two states suggest possible mechanisms of self-aggregation and silica precipitation.

Complex RNA structures are constructed from helical segments connected by flexible loops that move spontaneously and in response to binding of small molecule ligands and proteins. Understanding the conformational variability of RNA requires the characterization of the coupled time evolution of interconnected flexible domains. To elucidate the collective molecular motions and explore the conformational landscape of the HIV-1 TAR RNA, we describe a new methodology that utilizes energy-minimized structures generated by the program “Fragment Assembly of RNA with Full-Atom Refinement (FARFAR)”. We apply structural filters in the form of experimental residual dipolar couplings (RDCs) to select a subset of discrete energy-minimized conformers and carry out principal component analyses (PCA) to corroborate the choice of the filtered subset. We use this subset of structures to calculate solution T1 and T1ρrelaxation times for 13C spins in multiple residues in different domains of the molecule using two simulation protocols that we previously published. We match the experimental T1 times to within 2% and the T1ρ times to within less than 10% for helical residues. These results introduce a protocol to construct viable dynamic trajectories for RNA molecules that accord well with experimental NMR data and support the notion that the motions of the helical portions of this small RNA can be described by a relatively small number of discrete conformations exchanging over time scales longer than 1 μs.

Nature has evolved sophisticated strategies for engineering hardtissues through the interaction of proteins, and ultimately cells, with inorganic mineral phases. This process, called biomineralization, is how living organisms transform inorganic materials such as hydroxyapatite, calcite, and silica into highly intricate and organized structures. The remarkable material properties of shell, bone, and teeth come from the activities of proteins that function at the organic–inorganic interface. A better understanding of the biomolecular mechanisms used to promote or retard the formation of mineral-based structures could provide important design principles for the development of calcification inhibitors and promoters in orthopedics, cardiology, urology, and dentistry. With the knowledge of the structural basis for control of hard tissue growth by proteins, scientists could potentially develop materials using biomimetic principles with applications in catalysis, biosensors, electronic devices, and chromatographic separations, to name a few. Additionally, biomineralization also has potential applications in electronics, catalysis, magnetism, sensory devices, and mechanical design. Where man-made hard materials require the use of extreme temperatures, high pressure, and pH, biological organisms can accomplish these feats at ambient temperature and at physiological pH.

Despite the fact that many researchers want to identify and control the structure of proteins at material and biomineral interfaces, there is a decided lack of molecular-level structure information available for proteins at biomaterial interfaces in general. In particular, this holds for mammalian proteins that directly control calcification processes in hard tissue. The most fundamental questions regarding the secondary and tertiary structures of proteins adsorbed to material surfaces, how proteins catalyze the formation of biomineral composites, or how proteins interact at biomaterial interfaces remain unanswered. This is largely due to a lack of methods capable of providing high-resolution structural information for proteins adsorbed to material surfaces under physiologically relevant conditions.

In this Account, we highlight recent work that is providing insight into the structure and crystal recognition mechanisms of a salivary protein model system, as well as the structure and interactions of a peptide that catalyzes the formation of biosilica composites. To develop a better understanding of the structure and interactions of proteins in biomaterials, we have used solid-state NMR techniques to determine the molecular structure and dynamics of proteins and peptides adsorbed onto inorganic crystal surfaces and embedded within biomineral composites. This work adds to the understanding of the structure and crystal recognition mechanisms of an acidic human salivary phosphoprotein, statherin.

Nature has evolved sophisticated strategies for engineering hard tissues through the interaction of proteins, and ultimately cells, with inorganic mineral phases. The remarkable material properties of shell, bone and teeth thus result from the activities of proteins that function at the organic-inorganic interface. A better understanding of the biomolecular mechanisms used to promote or retard the formation of mineral-based structures could provide important design principles for the development of calcification inhibitors and promoters in orthopedics, cardiology, urology, and dentistry. In addition to investigating the molecular-level basis for the recognition of biomineral surfaces and the control of hard tissue growth by proteins, the development of materials using biomimetic principles has potential applications in catalysis, biosensors, electronic devices, chromatographic separations, to name only a few.

Despite the high level of interest in elucidating and controlling the structure of proteins at material and biomineral interfaces, there is a decided lack of molecular-level structure information available for proteins at biomaterial interfaces in general, and in particular for mammalian proteins that directly control calcification processes in hard tissue. The most fundamental questions regarding the secondary and tertiary structures of proteins adsorbed to material surfaces, how proteins catalyze the formation of biomineral composites, or how proteins interact at biomaterial interfaces, remain unanswered, largely due to a lack of methods capable of providing high resolution structural information for proteins adsorbed to material surfaces under physiologically relevant conditions (i.e. fully hydrated).

In order to develop a better understanding of the structure and interactions of proteins in biomaterials, we have begun to utilize solid-state NMR techniques to determine the molecular structure and dynamics of proteins and peptides on inorganic crystal surfaces and within biomineral composites. In this review, we will highlight recent work that is providing insight into the structure and crystal recognition mechanisms of a salivary protein model system, as well as the structure and interactions of a peptide which catalyzes the formation of biosilica composites.

Solid state NMR can provide detailed structural and dynamic information on biological systems that cannot be studied under solution conditions, and can investigate motions which occur with rates that cannot be fully studied by solution NMR. This approach has successfully been used to study proteins, but the application of multidimensional solid state NMR to RNA has been limited because reported line widths have been too broad to execute most multidimensional experiments successfully. A reliable method to generate spectra with narrow line widths is necessary to apply the full range of solid state NMR spectroscopic approaches to RNA. Using the HIV-1 transactivation response (TAR) RNA as a model, we present an approach based on precipitation with polyethylene glycol that improves the line width of (13)C signals in TAR from >6 ppm to about 1 ppm, making solid state 2D NMR studies of selectively enriched RNAs feasible at ambient temperature.

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein–surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.

Solution NMR spectroscopy can elucidate many features of the structure and dynamics of macromolecules, yet relaxation measurements, the most common source of experimental information on dynamics, can sample only certain ranges of dynamic rates. A complete characterization of motion of a macromolecule thus requires the introduction of complementary experimental approaches. Solid-state NMR spectroscopy successfully probes the time scale of nanoseconds to microseconds, a dynamic window where solution NMR results have been deficient, and probes conditions where the averaging effects of rotational diffusion of the molecule are absent. Combining the results of the two distinct techniques within a single framework provides greater insight into dynamics, but this task requires the common interpretation of results recorded under very different experimental conditions. Herein, we provide a unified description of dynamics that is robust to the presence of large-scale conformational exchange, where the diffusion tensor of the molecule varies on a time scale comparable to rotational diffusion in solution. We apply this methodology to the HIV-1 TAR RNA molecule, where conformational rearrangements are both substantial and functionally important. The formalism described herein is of greater generality than earlier combined solid-state/solution NMR interpretations, if detailed molecular structures are available, and can offer a more complete description of RNA dynamics than either solution or solid-state NMR spectroscopy alone.

Formation of the complex between human immunodeficiency virus type-1 Tat protein and the transactivation response region (TAR) RNA is vital for transcriptional elongation, yet the structure of the Tat-TAR complex remains to be established. The NMR structures of free TAR, and TAR bound to Tat-derived peptides have been obtained by solution NMR, but only a small number of intermolecular NOEs could be identified unambiguously, preventing the determination of a complete structure. Here we show that a combination of multiple solid state NMR REDOR experiments can be used to obtain multiple distance constraints from N to C spins within the backbone and side chain guanidinium groups of arginine in a Tat-derived peptide, using F spins incorporated into the base of U23 in TAR and P spins in the P22 and P23 phosphate groups. Distances between the side chain of Arg52 and the base and phosphodiester backbone near U23 measured by REDOR NMR are comparable to distances observed in solution NMR-derived structural models, indicating that interactions of TAR RNA with key amino acid side chains in Tat are the same in the amorphous solid state as in solution. This method is generally applicable to other protein-RNA complexes where crystallization or solution NMR has failed to provide high resolution structural information.

LKα14 is a 14 amino acid peptide with a periodic sequence of leucine and lysine residues consistent with an amphipathic α-helix. This “hydrophobic periodicity” has been found to result in an α-helical secondary structure at air–water interfaces and on both polar and nonpolar solid polymer surfaces. In this paper, the dynamics of LKα14 peptides, selectively deuterated at a single leucine and adsorbed onto polystyrene and carboxylated polystyrene beads, are studied using 2H magic angle spinning (MAS) solid state NMR over a 100 °C temperature range. We first demonstrate the sensitivity enhancement possible with 2H MAS techniques, which in turn enables us to obtain high-quality 2H NMR spectra for selectively deuterated peptides adsorbed onto solid polymer surfaces. The extensive literature shows that the dynamics of leucine side chains are sensitive to the local structural environment of the protein. Therefore, the degree to which the dynamics of leucine side chains and the backbone of the peptide LKα14 are influenced by surface proximity and surface chemistry is studied as a function of temperature with 2H MAS NMR. It is found that the dynamics of the leucine side chains in LKα14 depend strongly upon the orientation of the polymer on the surface, which in turn depends on whether the LKα14 peptide adsorbs onto a polar or nonpolar surface. 2H MAS line shapes therefore permit probes of surface orientation over a wide temperature range.

The complex of the HIV TAR RNA with the viral regulatory protein Tat is of considerable interest, but the plasticity of this interaction has made it impossible so far to establish the structure of that complex. In order to explore a new approach to obtain structural information on protein−RNA complexes, we performed 13C/15N−19F REDOR NMR experiments in the solid state on TAR bound to a peptide comprising the RNA-binding section of Tat. A critical arginine in the peptide was uniformly 13C and 15N labeled, and 5-fluorouridine was incorporated at the U23 position of TAR. REDOR irradiation resulted in dephasing of the 13C and 15N resonances, indicating the proximity of the U23(5F)−C and U23(5F)−N spin pairs. Best fits to the REDOR data show the U23(5F)−C distances and the U23(5F)−N distances are in good agreement with the distances obtained from solution NMR structures of partial complexes of Tat with TAR. These results demonstrate that it is possible to study protein−RNA complexes using solid-state REDOR NMR measurements, adding to a growing list of solid state techniques for studying protein−nucleic acid complexes.

Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized.

Functional RNA molecules are conformationally dynamic and sample a multitude of dynamic modes over a wide range of frequencies. Thus, a comprehensive description of RNA dynamics requires the inclusion of a broad range of motions across multiple dynamic rates which must be derived from multiple spectroscopies. Here we describe a slow conformational exchange theoretical approach to combining the description of local motions in RNA that occur in the ns-μs window and are detected by solid-state NMR with non-rigid rotational motion of the HIV-1 TAR RNA in solution as observed by solution NMR. This theoretical model unifies the experimental results generated by solution and solid-state NMR and provides a comprehensive view of the dynamics of HIV-1 TAR RNA, a well-known paradigm of an RNA where function requires extensive conformational rearrangements. This methodology provides a quantitative atomic level view of the amplitudes and rates of the local and collective displacements of the TAR RNA molecule, and provides directly motional parameters for the conformational capture hypothesis of this classical RNA-ligand interaction.

RNA and DNA can adopt highly different conformations to facilitate protein or ligand binding. These conformational changes are significant, since they often are an essential part of nucleic acid function. As a result, there is ever increasing interest in studying dynamics of nucleic acids and their connection to function. Here, we review NMR methods, in both the solid and liquid states, to study nucleic acid dynamics, including relaxation, residual dipolar couplings and lineshape analysis.

The power of combining sum frequency generation (SFG) vibrational spectroscopy and solid-state nuclear magnetic resonance (ssNMR) spectroscopy to quantify, with site specificity and atomic resolution, the orientation and dynamics of side chains in synthetic model peptides adsorbed onto polystyrene (PS) surfaces is demonstrated in this study. Although isotopic labeling has long been used in ssNMR studies to site-specifically probe the structure and dynamics of biomolecules, the potential of SFG to probe side chain orientation in isotopically labeled surface-adsorbed peptides and proteins remains largely unexplored. The 14 amino acid leucine-lysine peptide studied in this work is known to form an α-helical secondary structure at liquid-solid interfaces. Selective, individual deuteration of the isopropyl group in each leucine residue was used to probe the orientation and dynamics of each individual leucine side chain of LKα14 adsorbed onto PS. The selective isotopic labeling methods allowed SFG analysis to determine the orientations of individual side chains in adsorbed peptides. Side chain dynamics were obtained by fitting the deuterium ssNMR line shape to specific motional models. Through the combined use of SFG and ssNMR, the dynamic trends observed for individual side chains by ssNMR have been correlated with side chain orientation relative to the PS surface as determined by SFG. This combination provides a more complete and quantitative picture of the structure, orientation, and dynamics of these surface-adsorbed peptides than could be obtained if either technique were used separately.

Organisms use proteins such as statherin to control the growth of hydroxyapatite (HAP), which is the principal component of teeth and bones. Though much emphasis has been placed on the acidic character of these proteins, the role of their basic amino acids is not well understood. In this work, solid state nuclear magnetic resonance was used to probe the interaction of the basic arginine side chains with the HAP surface. Statherin samples were individually labeled at each arginine site, and the distance to the surface was measured using the Rotational Echo DOuble Resonance (REDOR) technique. The results indicate a strong coupling between the R9 and R10 residues and the phosphorus atoms on the surface, with internuclear distances of 4.62 ± 0.29 Å and 4.53 ± 0.16 Å, respectively. Conversely, results also indicate weak coupling between R13 and the surface, suggesting this residue is more removed from the surface than R9 and R10. Combining these results with previous data, a new model for the molecular recognition of HAP by statherin is constructed.

The last few years have seen a remarkable increase in interest in the role of protein motion in catalysis, the energetics of protein folding, and molecular recognition. While motion by conformational adaptation is also very important for nucleic acids (many RNAs and DNAs function by undergoing large conformational changes in response to binding of a protein or small molecule), it is still not clear how motion contributes to the function of nucleic acids. There is a clear need to study motions and conformational transitions of nucleic acids by applying biophysical techniques that extend the description of DNA and RNA beyond the familiar static structures. It is well known that different dynamic spectroscopies display variable sensitivity to different rates of motion; therefore, if we rely upon any single type of spectroscopic measurement we run the risk of obtaining either an incomplete or incorrect description of internal molecular motions. Here, we use solution and solid-state NMR to study the dynamics for two paradigmatic nucleic acid systems whose biological function depends on their ability to change structure: the flipping out of a deoxycytidine by the HhaI methyltransferase and TAR RNA which must undergo a structural rearrangement of its bulged loop to bind to the tat protein. In both cases, structures in the absence and presence of ligand are well described. Yet, little experimental data exists to define over the dynamic pathways linking these states, and how they depend on sequence. In both cases, we use solid-state 2H NMR line shapes to probe for the presence of internal motions in the microsecond to nanaosecond timescales and relaxation to investigate dynamics at nanoseconds and shorter. We show that solid-state NMR can quantify motions at timescales not easily probed by solution NMR relaxation techniques and we show that solution and solid-state NMR views of the internal and overall rotations of these molecules can produce a unified view of the dynamics of these biomolecular systems.

Many RNAs undergo large conformational changes in response to the binding of proteins and small molecules. However, when RNA functional dynamics occur in the nanosecond-microsecond time scale, they become invisible to traditional solution NMR relaxation methods. Residual dipolar coupling methods have revealed the presence of extensive nanosecond-microsecond domain motions in HIV-1 TAR RNA, but this technique lacks information on the rates of motions. We have used solid-state deuterium NMR to quantitatively describe trajectories of key residues in TAR by exploiting the sensitivity of this technique to motions that occur in the nanosecond-microsecond regime. Deuterium line shape and relaxation data were used to model motions of residues within the TAR binding interface. The resulting motional models indicate two functionally essential bases within the single-stranded bulge sample both the free and Tat-bound conformations on the microsecond time scale in the complete absence of the protein. Thus, our results strongly support a conformational capture mechanism for recognition: the protein does not induce a new RNA structure, but instead captures an already-populated conformation.

Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases, a strong NH mode near 3300 cm^-1 is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain, since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode, we studied¹⁵N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an α-helical secondary structure (LKα14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. ¹⁵N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm-1 on SiO₂ and 13 cm^-1 on CaF₂. This clearly shows the 3300 cm^-1 NH feature is associated with side chain NH stretches and not with backbone amide modes.

The side chain carboxyl groups of acidic proteins found in the extra-cellular matrix (ECM) of mineralized tissues play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), the principal mineral component of bone and teeth. Among the acidic proteins found in the saliva is statherin, a 43-residue tyrosine-rich peptide that is a potent lubricant in the salivary pellicle and an inhibitor of both HAP crystal nucleation and growth. Three acidic amino acids-D1, E4, and E5-are located in the N-terminal 15 amino acid segment, with a fourth amino acid, E26, located outside the N-terminus. We have utilized ¹³C{³¹P} REDOR NMR to analyze the role played by acidic amino acids in the binding mechanism of statherin to the HAP surface by measuring the distance between the δ-carboxyl ¹³C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and ³¹P spins of the phosphate groups at the HAP surface. ¹³C{³¹P} REDOR studies of glutamic-5-¹³C acid incorporated at positions E4 and E26 indicate a ¹³C-³¹P distance of more than 6.5 Åbetween the side chain carboxyl ¹³C spin of E4 and the closest ³¹P in the HAP surface. In contrast, the carboxyl ¹³C spin at E5 has a much shorter ¹³C-³¹P internuclear distance of 4.25 +/- 0.09 Å, indicating that the carboxyl group of this side chain interacts directly with the surface. ¹³C T1ρ and slow-spinning MAS studies indicate that the motions of the side chains of E4 and E5 are more restricted than that of E26. Together, these results provide further insight into the molecular interactions of statherin with HAP surfaces.

The artificial amphiphilic peptide LKα14 adopts a helical structure at interfaces, with opposite orientation of its leucine (L, hydrophobic) and lysine (K, hydrophilic) side chains. When peptides are adsorbed onto surfaces, different residue side chains necessarily have different proximities to the surface, depending on both their position in the helix and the composition of the surface itself. Deuterating the individual leucine residues (isopropyl-d₇) permits the use of solid-state deuterium NMR spectroscopy as a site-specific probe of side-chain dynamics. In conjunction with sum-frequency generation as a probe of the peptide-binding face, we demonstrate that the mobility of specific leucine side chains at the interface is quantifiable in terms of their surface proximity.

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms-ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in ²H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state ¹³C relaxation measurements, we establish that ns-μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.

Both solid-state and solution NMR relaxation measurements are routinely used to quantify the internal dynamics of biomolecules, but in very few cases have these two techniques been applied to the same system, and even fewer attempts have been made so far to describe the results obtained through these two methods through a common theoretical framework. We have previously collected both solution ¹³C and solid-state ²H relaxation measurements for multiple nuclei within the furanose rings of several nucleotides of the DNA sequence recognized by HhaI methyltransferase. The data demonstrated that the furanose rings within the GCGC recognition sequence are very flexible, with the furanose rings of the cytidine, which is the methylation target, experiencing the most extensive motions. To interpret these experimental results quantitatively, we have developed a dynamic model of furanose rings based on the analysis of solid-state ²H line shapes. The motions are modeled by treating bond reorientations as Brownian excursions within a restoring potential. By applying this model, we are able to reproduce the rates of ²H spin-lattice relaxation in the solid and¹³C spin-lattice relaxation in solution using comparable restoring force constants and internal diffusion coefficients. As expected, the ¹³C relaxation rates in solution are less sensitive to motions that are slower than overall molecular tumbling than to the details of global molecular reorientation, but are somewhat more sensitive to motions in the immediate region of the Larmor frequency. Thus, we conclude that the local internal motions of this DNA oligomer in solution and in the hydrated solid state are virtually the same, and we validate an approach to the conjoint analysis of solution and solid-state NMR relaxation and line shapes data, with wide applicability to many biophysical problems.

A nucleoside carrying a perfluorinated tert-butyl group (4) was prepared by a Sonogashira coupling of 5-iodo-2′-deoxyuridine with 4,4,4-trifluoro-3,3-bis(trifluoromethyl)-1-butyne in nearly quantitative yield and subsequently incorporated into DNA oligomers. Thermal denaturation studies showed that 4 had a negligible effect on duplex stabilty when compared to thymidine. Transition from single strand to duplex was monitored by ¹⁹F NMR spectroscopy at micromolar concentrations of oligomers, demonstrating the sensitivity of 4 as an NMR reporter nucleoside.

The dynamics of the phosphodiester backbone in the [5′-GCGC-3′]2 moiety of the DNA oligomer [d(G1A2T3A4G5C6G7C8T9A10T11C12)]2 are studied using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs nonstereospecifically deuterated on the 5′ methylene group of nucleotides within the [5′-GCGC-3′]2 moiety indicated that all of these positions are structurally flexible. Previous work has shown that methylation reduces the amplitude of motion in the phosphodiester backbone and furanose ring of the same DNA, and our observations indicate that methylation perturbs backbone dynamics through not only a loss of mobility but also a change of direction of motion. These NMR data indicate that the [5′-GCGC-3′]2 moiety is dynamic, with the largest amplitude motions occurring nearest the methylation site. The change of orientation of this moiety in DNA upon methylation may make the molecule less amenable to binding to the HhaI endonuclease.

The dynamics of the furanose rings in the GCGC moiety of the DNA oligomer [d(G1A2T3A4G5C6G7C8T9A10T11C12)]2 are studied by using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs selectively deuterated on the furanose rings of nucleotides within the 5′-GCGC-3′ moiety indicated that all of these positions are structurally flexible. The furanose ring within the deoxycytidine that is the methylation target displays the largest-amplitude structural changes according to the observed deuterium NMR line shapes, whereas the furanose rings of nucleotides more remote from the methylation site have less-mobile furanose rings (i.e., with puckering amplitudes < 0.3 A). Previous work has shown that methylation reduces the amplitude of motion in the phosphodiester backbone of the same DNA, and our observations indicate that methylation perturbs backbone dynamics through the furanose ring. These NMR data indicate that the 5′-GCGC-3′ is dynamic, with the largest-amplitude motions occurring nearest the methylation site. The inherent flexibility of this moiety in DNA makes the molecule more amenable to the large-amplitude structural rearrangements that must occur when the DNA binds to the HhaI methyltransferase.

Solution and solid-state NMR measurements were used together to examine motion in three sites in the HIV-1 TAR RNA. We wished to investigate the dynamics facilitating the conformational rearrangements the TAR RNA must undergo for Tat binding and in particular to characterize the full range of motional time scales accessible to this RNA. Our results demonstrate that the dynamics in TAR involving residues essential to Tat binding include not only the faster motions detected by solution relaxation measurements but also a significant component in the us-ns time scale.

Proteins are found to be involved in interaction with solid surfaces in numerous natural events. Acidic proteins that adsorb to crystal faces of a biomineral to control the growth and morphology of hard tissue are only one example. Deducing the mechanisms of surface recognition exercised by proteins has implications to osteogenesis, pathological calcification and other proteins functions at their adsorbed state. Statherin is an enamel pellicle protein that inhibits hydroxyapatite nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. Here, we highlight some of the insights we obtained recently using both thermodynamic and solid state NMR measurements to the adsorption process of statherin to hydroxyapatite. We combine macroscopic energy characterization with microscopic structural findings to present our views of protein adsorption mechanisms and the structural changes accompanying it and discuss the implications of these studies to understanding the functions of the protein adsorbed to the enamel surfaces.

Recognition of RNA by proteins and small molecules often involves large changes in RNA structure and dynamics, yet very few studies have so far characterized these motional changes. Here we extend to the protein-bound RNA recent ¹³C relaxation studies of motions in the RNA recognized by human U1A protein, a well-known model for protein-RNA recognition. Changes in relaxation observed upon complex formation demonstrate that the protein-binding site becomes rigid in the complex, but the upper stem-loop that defines the secondary structure of this RNA experiences unexpected motional freedom. By using a helix elongation strategy, we observe that the upper stem-loop moves independently of the remainder of the structure also in the absence of U1A. Surprisingly, RNA residues making important intermolecular contacts in the structure of the complex exhibit increased flexibility in the presence of the protein. Both of these results support the hypothesis that RNA-binding proteins select a structure that optimizes intermolecular contacts in the manifold of conformations sampled by the free RNA and that protein binding quenches these motions. Together with previous studies of the RNA-bound protein, they also demonstrate that protein-RNA interfaces experience complex motions that modulate the strength of individual interactions.

Salivary statherin is a highly acidic, 43 amino acid residue protein that functions as an inhibitor of primary and secondary crystallization of the biomineral hydroxyapatite. The acidic domain at the N-terminus was previously shown to be important in the binding of statherin to hydroxyapatite surfaces. This acidic segment is followed by a basic segment whose role is unclear. In this study, the role of the basic amino acids in the hydroxyapatite adsorption thermodynamics has been determined using isothermal titration calorimetry and equilibrium adsorption isotherm analysis. Single point mutations of the basic side chains to alanine lowered the binding affinity to the surface but did not perturb the maximal surface coverage and the adsorption enthalpy. The structural and dynamic properties of the single point mutants as characterized by solid-state NMR techniques were not altered either. Simultaneous replacement of all four basic amino acids with alanine lowered the adsorption equilibrium constant by 5-fold and the maximal surface coverage by nearly 2-fold. The initial exothermic phase of adsorption exhibited by native statherin is preserved in this mutant, along with the alpha-helical structure and the dynamic properties of the N-terminal domain. These results help to refine the two binding site model of statherin adsorption proposed earlier in our study of wild-type statherin (Goobes, R., Goobes, G., Campbell, C.T., and Stayton, P.S. (2006) Biochemistry 45, 5576-5586). The basic charges function to reduce protein-protein charge repulsion on the HAP surface, and in their absence, there is a considerable decrease in statherin packing density on the surface at binding saturation.

Solution and solid-state NMR have been used conjointly to probe the internal motions of a DNA dodecamer containing the recognition site for the HhaI methyltransferase. The results strongly suggest that ns-us motions contribute to the functionally relevant dynamic properties of nucleic acids during DNA methylation.

Statherin is an enamel pellicle protein that inhibits hydroxyapatite (HAP) nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. We report here from solid-state NMR measurements that the protein’s C-terminal region folds into an alpha-helix upon adsorption to HAP crystals. This region contains the binding sites for bacterial fimbriae that mediate bacterial cell adhesion to the surface of the tooth. The helical segment is shown through long-range distance measurements to fold back onto the intermediate region (residues Y16–P28) defining the global fold of the protein. Statherin, previously shown to be unstructured in solution, undergoes conformation selection on its substrate mineral surface. This surface-induced folding of statherin can be related to its functionality in inhibiting HAP crystal growth and can explain how oral pathogens selectively recognize HAP-bound statherin.

Acidic proteins found in mineralized tissues act as nature’s crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. ¹⁵N³¹P rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed ¹⁵N REDOR decay. We performed ³¹P-³¹P recoupling experiments in HAP and (NH₄)₂HPO₄ (DHP) to explore the nature of dipolar coupled ³¹P spin networks. These studies indicate that extensive networks of dipolar coupled ³¹P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out ¹⁵N³¹P REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended ³¹P coupled spin network simulates the REDOR data well, a simplified ³¹P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The ³¹P-³¹P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two ³¹P spins. We incorporated this coupling and applied the above approach to reanalyze the ¹⁵N³¹P REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain ¹⁵N-³¹P distances between 3.3 and 5 Å from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.

Extracellular matrix proteins regulate hard tissue growth by acting as adhesion sites for cells, by triggering cell signaling pathways, and by directly regulating the primary and/or secondary crystallization of hydroxyapatite, the mineral component of bone and teeth. Despite the key role that these proteins play in the regulation of hard tissue growth in humans, the exact mechanism used by these proteins to recognize mineral surfaces is poorly understood. Interactions between mineral surfaces and proteins very likely involve specific contacts between the lattice and the protein side chains, so elucidation of the nature of interactions between protein side chains and their corresponding inorganic mineral surfaces will provide insight into the recognition and regulation of hard tissue growth. Isotropic chemical shifts, chemical shift anisotropies (CSAs), NMR line-width information, ¹³C rotating frame relaxation measurements, as well as direct detection of correlations between ¹³C spins on protein side chains and ³¹P spins in the crystal surface with REDOR NMR show that, in the peptide fragment derived from the N-terminal 15 amino acids of salivary statherin (i.e., SN-15), the side chain of the phenylalanine nearest the C-terminus of the peptide (F14) is dynamically constrained and oriented near the surface, whereas the side chain of the phenylalanine located nearest to the peptide’s N-terminus (F7) is more mobile and is oriented away from the hydroxyapatite surface. The relative dynamics and proximities of F7 and F14 to the surface together with prior data obtained for the side chain of SN-15’s unique lysine (i.e., K6) were used to construct a new picture for the structure of the surface-bound peptide and its orientation to the crystal surface.

Magic angle spinning NMR techniques can be used to determine the molecular structure of proteins adsorbed onto polymer and mineral surfaces, but the degree to which the orientation of proteins on surfaces can be uniquely determined by NMR is less well understood. In this manuscript, REDOR data obtained from model systems are analyzed with a view to determine the orientation of rare spins coupled to a lattice populated by strongly coupled spin Click to view the MathML source nuclei. When the surface is populated by closely spaced spins, the REDOR dephasing of a rare spin on the protein contact point to the surface is under certain circumstances complicated by contributions from homonuclear dipolar interactions between the spins of the lattice. To study multiple spin effects on the dephasing signal in rotational-echo-double-resonance experiments, we carried out a measurement on crystalline diammonium hydrogen phosphate as a model for a spin system with multiple dipolar interactions. Information about the ³¹P–³¹P interactions is gathered from the reference measurement in the experiment. To fit the experimental ¹⁵N and ³¹P dephasing data well, it was necessary to account for as many as 6 and 8 spins in simulations, respectively. Using a single spin-pair interaction with an unknown distance yielded a good fit to the ³¹P data with a distance of 2.7 Å that is nearly an Angström shorter than the shortest distance in the crystal structure. Homonuclear couplings are shown to have a significant effect on the expected dephasing.

REDOR is a solid-state NMR technique frequently applied to biological structure problems. Through incorporation of phosphorothioate groups in the nucleic acid backbone and mono-fluorinated nucleotides, ³¹P¹⁹F REDOR has been used to study the binding of DNA to drugs and RNA to proteins through the detection of internuclear distances as large as 13–14 Å. In this work, ³¹P¹⁹F REDOR is further refined for use in nucleic acids by the combined use of selective placement of phosphorothioate groups and the introduction of nucleotides containing trifluoromethyl (–CF3) groups. To ascertain the REDOR-detectable distance limit between an unique phosphorous spin and a trifluoromethyl group and to assess interference from intermolecular couplings, a series of model compounds and DNA dodecamers were synthesized each containing a unique phosphorous label and trifluoromethyl group or a single ¹⁹F nucleus. The dipolar coupling constants of the various ³¹P and ¹⁹F or –CF3 containing compounds were compared using experimental and theoretical dephasing curves involving several models for intermolecular interactions.

Hydroxyapatite (HAP) is the main mineral component of teeth. It is well-known that several salivary proteins and peptides bind strongly to HAP to regulate crystal growth. Interactions between a peptide derived from the N-terminal fragment of the salivary protein statherin and HAP were measured utilizing rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR). The REDOR measurement from the side chain of the salivary peptide to the HAP surface is complicated by two effects: a possible additional dipolar coupling to a phosphorylated side chain and the potential proximity of phosphorus atoms to each other, resulting in a homonuclear dipolar interaction. Both of these effects were addressed, and the smallest model applicable to our system includes the nitrogen-15 (¹⁵N) spin in the lysine side chain and two phosphorus-31 (³¹P) spins, at least one of which must be from the surface phosphates of the HAP.

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 Å decrease in the separation between phosphorothioate and 2’F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR–tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, ³¹P–¹⁹F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA–peptide complex.

In this paper we describe solid-state NMR experiments that provide information on the structures of surface-immobilized peptides. The peptides are covalently bound to alkanethiolates that are self-assembled as monolayers on colloidal gold nanoparticles. The secondary structure of the immobilized peptides was characterized by quantifying the Ramachandran angles and . These angles were determined in turn from distances between backbone carbonyl ¹³C spins, measured with the double-quantum filtered dipolar recoupling with a windowless sequence experiment, and by determination of the mutual orientation of chemical shift anisotropy tensors of ¹³C carbonyl spins on adjacent peptide planes, obtained from the double-quantum cross-polarization magic-angle spinning spectrum. It was found that peptides composed of periodic sequences of leucines and lysines were bound along the length of the peptide sequence and displayed a tight alpha-helical secondary structure on the gold nanoparticles. These results are compared to similar studies of peptides immobilized on hydrophobic surfaces.

We have performed solid-state ³¹P-¹⁹F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA2’FUTGGC)·d(GCCAAT(pS)TT GCG) (A3T2) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the 2’FU7–pS19 inter-label distance, was 9.4 ± 0.7 Å, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 Å decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 Å, consistent with the results of a previous solution NMR study of the 2:1 complex. These ³¹P-¹⁹F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7–14 Å in DNA–drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.

Proteins found in mineralized tissues act as nature’s crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (bones/teeth) and calcium oxalate (kidney stones). Despite their importance in hard-tissue formation and remodeling, and in pathological processes such as stone formation and arterial calcification, there is little known of the protein structure-function relationships that govern hard-tissue engineering. Here we review early studies that have utilized solid-state NMR (ssNMR) techniques to provide in situ secondary-structure determination of statherin and statherin peptides on their biologically relevant hydroxyapatite (HAP) surfaces. In addition to direct structural study, molecular dynamics studies have provided considerable insight into the protein-binding footprint on hydroxyapatite. The molecular insight provided by these studies has also led to the design of biomimetic fusion peptides that utilize nature’s crystal-recognition mechanism to display accessible and dynamic bioactive sequences from the HAP surface. These peptides selectively engage adhesion receptors and direct specific outside-in signaling pathway activation in osteoblast-like cells.

Keywords: Biomineralization, molecular recognition, hydroxyapatite, biomaterials

Dipolar recoupling pulse sequences are of great importance in magic angle spinning solid-state NMR. Recoupling sequences are used for excitation of double-quantum coherence, which, in turn, is employed in experiments to estimate internuclear distances and molecular torsion angles. Much effort is spent on the design of recoupling sequences that are able to produce double-quantum coherence with high efficiency in demanding spin systems, i.e., spin systems with small dipole-dipole couplings and large chemical-shift anisotropies (CSAs). The sequence should perform robustly under a variety of experimental conditions. This paper presents experiments and computer calculations that extend the theory of double-quantum coherence preparation from the strong coupling/small CSA limit to the weak coupling limit. The performance of several popular dipole-dipole recoupling sequences-DRAWS, POST-C7, SPC-5, R1, and R2-are compared. It is found that the optimum performance for several of these sequences, in the weak coupling/large CSA limit, varies dramatically, with respect to the sample spinning speed, the magnitude and orientation of the CSAs, and the magnitude of dipole-dipole couplings. It is found that the efficiency of double-quantum coherence preparation by -encoded sequences departs from the predictions of first-order theory. The discussion is supported by density-matrix calculations.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composites technologies. Here, we describe both the theory and practice of double-quantum solid-state NMR (ssNMR) structure-determination techniques, as they are used to determine the secondary structures of surface-adsorbed peptides and proteins. In particular, we have used ssNMR dipolar techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Here, we also review NMR data on peptides designed to adsorb from aqueous solutions onto highly porous hydrophobic surfaces with specific helical secondary structures. The adsorption or covalent attachment of biological macromolecules onto polymer materials to improve their biocompatibility has been pursued using a variety of approaches, but key to understanding their efficacy is the verification of the structure and dynamics of the immobilized biomolecules using double-quantum ssNMR spectroscopy.

The adsorption or covalent attachment of biological macromolecules onto polymer materials to improve their biocompatibility has been pursued using a variety of approaches, but key to understanding their efficacy is the verification of the structure and dynamics of the immobilized biomolecules. Here we present data on peptides designed to adsorb from aqueous solutions onto highly porous hydrophobic surfaces with specific helical secondary structures. Small linear peptides composed of alternating leucine and lysine residues were synthesized, and their adsorption onto porous polystyrene surfaces was studied using a combination of solid-state NMR techniques. Using conventional solid-state NMR experiments and newly developed double-quantum techniques, their helical structure was verified. Large-amplitude dynamics on the NMR time scale were not observed, suggesting irreversible adsorption of the peptides. Their association, adsorption, and structure were examined as a function of helix length and sequence periodicity, and it was found that, at higher solution concentrations, peptides as short as seven amino acids adsorb with defined secondary structures. Two-dimensional double-quantum experiments using ¹³C-enriched peptide sequences allow high-resolution determination of secondary structure in heterogeneous environments where the peptides are a minor component of the material. These results shed light on how polymeric surfaces may be surface-modified by structured peptides and demonstrate the level of molecular structural and dynamic information solid-state NMR can provide.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicate the N-terminus is a-helical in character with the residues directly binding to the HAP being stabilized in the a-helical conformation by the presence of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of statherin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain outside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.

Determination of the conformational flexibility of the furanose ring is of vital importance in understanding the structure of DNA. In this work we have applied a model of furanose ring motion to the analysis of deuterium line shape data obtained from sugar rings in solid hydrated DNA. The model describes the angular trajectories of the atoms in the furanose ring in terms of pseudorotation puckering amplitude (q) and the pseudorotation puckering phase . Fixing q, the motion is thus treated as Brownian diffusion through an angular-dependent potential U(). We have simulated numerous line shapes varying the adjustable parameters, including the diffusion coefficient D, pseudorotation puckering amplitude q, and the form of the potential U(). We have used several forms of the potential, including equal double-well potentials, unequal double-well potentials, and a potential truncated to “second order” in the Fourier series. To date, we have obtained best simulations for both equilibrium and nonequilibrium (partially relaxed) solid-state deuterium NMR line shapes for the sample [2′ ‘-²H]-2′-deoxycytidine at the position C3 (underlined) in the DNA sequence [d(CGCGAATTCGCG)]2, using a double-well potential with an equal barrier height of U0 = 5.5kBT ( 3.3 kcal/mol), a puckering amplitude of q = 0.4 Å, and a diffusion coefficient characterizing the underlying stochastic jump rate D = 9.9 × 108 Hz. Then the rate of flux for the C-D bond over the barrier, i.e., the escape velocity or the overall rate of puckering between modes, was found to be 0.7 × 107 Hz.

Base methylation plays an important role in numerous biological functions of DNA, from inhibition of cleavage by endonucleases to inhibition of transcription factor binding. Studies of nucleic acid structure have shown little differences in unmethylated DNAs and the identical sequence containing methylated analogues. We have investigated changes in the local dynamics of DNA upon substitution of a methylated cytosine analogue for cytosine using solid-state deuterium NMR. In particular, we have observed changes in the local dynamics at the target site of the M. HhaI restriction system. These studies observe changes in the amplitudes of the local backbone dynamics at the actual target site of the HhaI methyltransferase. This conclusion is another indication that the significant result of base methylation is to perturb the local dynamics, and therefore the local conformational flexibility, of the DNA helix, inhibiting or restricting the protein’s ability to manipulate the DNA helix in order to perform its chemical alterations.

Dipolar decoupling of protons with radiofrequency field amplitudes comparable to those used for the rare-spin refocusing and dephasing π pulses results in accurate, high-sensitivity determinations of internuclear distances in rotational-echo double-resonance experiments and simulations performed on ¹³C and ¹⁵N-labeled DL-alanine.

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha v melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha v subpopulation demonstrated that the alpha vbeta 3 integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.

Solid-state deuterium NMR is used to investigate perturbations of the local, internal dynamics in the EcoRI restriction binding site, -GAATTC- induced by cytidine methylation. Methylation of the cytidine base in this sequence is known to suppress hydrolysis by the EcoRI restriction enzyme. Previous solid-state deuterium NMR studies have detected large amplitude motions of the phosphate-sugar backbone at the AT-CG junction of the unmethylated DNA sequence. This study shows that methylation of the cytidine base in a CpG dinucleotide reduces the amplitudes of motions of the phosphate-sugar backbone. These observations suggest a direct link between suppression of the amplitudes of localized, internal motions of the sugar-phosphate backbone of the DNA and inhibition of restriction enzyme cleavage.

Proteins play an important role in inorganic crystal engineering during the development and growth of hard tissues such as bone and teeth. Although many of these proteins have been studied in the liquid state, there is little direct information describing molecular recognition at the protein-crystal interface. Here we have used ¹³C solid-state NMR (SSNMR) techniques to investigate the conformation of an N-terminal peptide of salivary statherin both free and adsorbed on hydroxyapatite (HAP) crystals. The torsion angle was determined at three positions along the backbone of the phosphorylated N-terminal 15 amino acid peptide fragment (DpSpSEEKFLRRIGRFG) by measuring distances between the backbone carbonyls carbons in the indicated adjacent amino acids using dipolar recoupling with a windowless sequence (DRAWS). Global secondary structure was determined by measuring the dipolar coupling between the ¹³C backbone carbonyl and the backbone ¹⁵N in the i i + 4 residues (DpSpSEEKFLRRIGRFG) using rotational echo double resonance (REDOR). Peptides singly labeled at amino acids pS3, L8, and G12 were used for relaxation and line width measurements. The peptides adsorbed to the HAP surface have an average of -85 at the N-terminus (pSpS), -60 in the middle (FL) and -73 near the C-terminus (IG). The average angle measured at the pSpS position and the observed high conformational dispersion suggest a random coil conformation at this position. However, the FL position displays an average that indicates significant -helical content, and the long time points in the DRAWS experiment fit best to a relatively narrow distribution of that falls within the protein data bank -helical conformational space. REDOR measurements confirm the presence of helical content, where the distance across the LG hydrogen bond of the adsorbed peptide has been found to be 5.0 Å. The angle measured at the IG position falls at the upper end of the protein data bank -helical distribution, with a best fit to a relatively broad distribution that is consistent with a distribution of -helix and more extended backbone conformation. These results thus support a structural model where the N-terminus is disordered, potentially to maximize interactions between the HAP surface and the negatively charged side chains found in this region, the middle portion is largely -helical, and the C-terminus has a more extended conformation (or a mixture of helix and extended conformations).

A combination of Solid State NMR (SSNMR) dipolar recoupling and double quantum CSA-CSA correlation experiments are used to determine the Ramachandran angles phi and psi in a crystalline tripeptide (AGG) and a 14 amino acid peptide designed to be helical. The advantage of the SSNMR approach described herein is the ability to measure both and to high resolution using a single noncrystalline, doubly carbonyl labeled peptide. It is also shown that DRAWS and DQDRAWS data are insensitive to 14N-¹³C and ¹⁵N-¹³C dipolar couplings making corrections for these effects unnecessary. Extremes in secondary structure (e.g., a-helix vs b-sheet) can be discerned by simple inspection of DQDRAWS spectra. Subtleties in secondary structure (a-helix vs 3-10-helix) can be distinguished by simulation of the DQDRAWS spectrum.

To systematically explore the effects of spin system size and geometry on the precision and accuracy of two-dimensional solid state NMR distance measurements, we have applied two homonuclear dipolar recoupling experiments, 2D DRAWS and 2D RFDR, to five polycrystalline samples of uniformly or selectively ¹³C-labeled cytidine. Distance information has been obtained from the intensities and time behavior of cross-peaks observed in the resulting two-dimensional spectra. The experimental cross-peak buildup curves obtained from these crystalline cytidine samples have been analyzed by comparison with simulations. In uniformly ¹³C-labeled cytidine, indirect coherence transfer mechanisms lead to low-precision distance measurements not unlike those measured in solution state NOESY experiments. In the selectively labeled cytidines, the distance measurements are considerably more precise, allowing the possibility of very accurate structure determinations from selectively or randomly labeled spin systems. Of the two techniques, the 2D DRAWS method allows identification of indirect coherence transfer mechanisms that hinder accurate distance measurement.

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two ¹³C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ± 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete alpha -helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an alpha -helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

A solid-state deuterium NMR study of localized mobility at the C9pG10 step in the DNA dodecamer [d(CGCGAATTCGCG)]2 is described. In contrast to the results of earlier deuterium NMR studies of furanose ring and backbone dynamics within the d(AATT) moiety, the furanose ring and helix backbone of dC9 display large amplitudes of motion on the 0.1 ms time scale at hydration levels characteristic of the B form structure. Solid-state deuterium NMR line shape data obtained from labeled dC9 DNA are interpreted using a composite motion model, in which the DNA oligomer is treated as rotating as a whole about the helix axis, while the base, furanose ring, and phosphodiester backbone execute localized motions. Consistent with past solid-state NMR studies, the amplitude and rate of the uniform rotation of the dC9-labeled oligomer are found to be sensitive to hydration level. Amplitudes of localized reorientational motions of C-D bonds in the furanose ring and backbone of dC9 are found to be larger than the librational amplitudes for the C-D bonds in the base of dC9, indicating that the pyrimidine base sugar does not move as a rigid entity and intersects a locally flexible region of the phosphodiester backbone. At hydration levels corresponding to 10-12 waters per nucleotide, Zeeman relaxation times for the furanose ring and backbone deuterons of dC9 in B form DNA equal 0.025 and 0.03 ms, respectively, and are the shortest relaxation times observed thus far for any deuteron in the DNA dodecamer at comparable hydration levels. The results of this solid-state NMR study suggest the existence of a significant dynamic component of sequence-specific recognition in this system.

Methods for the analysis and design of solid state nuclear magnetic resonance (NMR) probes are presented. These techniques have been applied to a 200 MHz (¹H Larmor frequency) ¹H–¹⁹F–¹³C cross polarization magic angle spinning probe. Transmission lines have been used throughout the design enabling the production of a highly power efficient probe (¹H-22 detecting ¹⁹F NMR signals while applying proton decoupling fields of 250 kHz. These techniques have proven very useful in designing the probe discussed within the text but are not confined to such a triple resonant design. In fact the techniques are sufficiently general to be useful to all radio frequency design efforts including deuterium wide-line probes and multiresonant high resolution NMR probes. A series of simple but useful design concepts are presented which are used to predict the efficiencies of the design presented. ©1998 American Institute of Physics.

We present a two-dimensional NMR technique for the measurement of dipolar couplings in polycrystalline solids. This experiment is fully transverse and uses a windowless dipolar recoupling pulse sequence (DRAWS, described in Gregory, D. M.; et al. Chem. Phys. Lett. 1995, 246, 654-663) to effect coherence transfer. Direct, internuclear coherence transfer produces negative cross-peaks in the 2D spectrum. Cross-peak development and experimental requirements for obtaining distances from the two-dimensional solid-state NMR spectra of two- and three-spin systems are discussed, and demonstrations are shown for thymidine-2,4-¹³C2 and L-alanine-¹³C3. Internuclear distances are derived by comparison of experimental cross-peak buildup curves with numerical simulations. In the three-spin system, indirect coherence-transfer mechanisms prohibit the interpretation of buildup curves as due to isolated spin pair interactions and limit the accuracy of some distance measurements. This 2D technique can also be used for spectral assignment, as demonstrated by an application to L-arginine·HCl-U-¹³C,¹⁵N.

A theoretical analysis of dipolar recoupling with a windowless multipulse irradiation (DRAWS) is presented. Analytical expressions that describe the degree to which the DRAWS pulse sequence recouples the dipolar interaction as a function of offset and spinning rate are derived using Floquet theory. Numerical methods are used to assess the performance of DRAWS in the preparation and detection of multiple quantum coherence. Simulations indicate that the mutual orientation of two or more CSA tensors can be obtained with high accuracy from double quantum spectra prepared and detected by DRAWS irradiation (DQDRAWS). These expectations are born out by experiment and in particular, the mutual orientation of three ¹³C CSA tensors in selectively labeled 2-deoxythymidine are determined from DQDRAWS data. The results of the DQDRAWS analysis of CSA tensor orientation in 2-deoxythymidine are shown to be in excellent agreement with results obtained by conventional methods. Using these CSA tensor orientations and an independent measurement of internuclear distance, a practical strategy is proposed and executed for deriving the mutual orientation of purine and pyrimidine bases in a DNA dodecamer from DQDRAWS data. The DQDRAWS method for determining the mutual orientation of rigid bodies in macromolecules is compared and contrasted to distance-based methods. ©1997 American Institute of Physics.

This chapter reviews the dynamics information obtained from experimental magnetic resonance studies of site-specifically labeled duplex DNA. A previous review (43) discusses the dynamics of duplex DNA; it develops a theory that shows how magnetic resonance experiments are used to detect those dynamics. The methods for obtaining information about dynamics as well as a summary of what is now known about the site-specific dynamics of DNA are presented. This review contains two methods sections which present results using electron paramagnetic resonance and nuclear magnetic resonance active probes.

Double quantum filtering in magic-angle-spinning (MAS) NMR experiments is used to correlate pairs of 18C nuclei in synthetically labelled DNA oligomers. A new pulse sequence is introduced for this purpose, DQ-DRAWS. This sequence is a modification of the DRAMA sequence (R. Tycko and G. Dabbagh, 1990, Chem. Phys. Lett., 173, 461). DQ-DRAWS is compared with earlier sequences and found to be more generally applicable to 18C nuclei with a large range of chemical shift anisotropies and offsets. Experimental results are presented for several small model compounds and for the DNA sequences [d(CGCGAA*T*TCGCG)]2 and [d(CGAGGT*T*TAAACCTCG)]2. The DNA molecules contain 18C labels on adjacent 2′-deoxythymidine bases. Applications of these techniques to the study of DNA-protein interactions and structure are discussed.

A windowless, homonuclear dipolar recoupling pulse sequence (DRAWS) is described and a theoretical basis for describing its recoupling performance is developed using numerical techniques. It is demonstrated that DRAWS recouples weak dipolar interactions over a broad range of experimental and molecular conditions. We discuss two spectroscopic control experiments, which help to take into account effects due to insufficient proton decoupling, relaxation, and static dipolar couplings to nearby ¹³C spins at natural abundance. Finally DRAWS is used in combination with selective ¹³C labeling to measure¹³C-¹³C distances in five doubly labeled DNA dodecamers, [d(CGCGAAT*T* CGCG)]2, which contain the binding site for the restriction enzyme EcoRI. The longest distance reported is 4.8 angstroms. In most cases the distances agree well with those derived from X-ray crystallographic data, although small changes in hydration level can result in relatively large changes in internuclear distances.

At TpA steps in DNA, the adenine base experiences exceptionally large amplitude (20-50) and slow (10 ms-1 us) motion [Kennedy et al. (1993) Biochemistry 32, 8022-8035] which has been correlated with transitions between multiple conformational states [Lefevre et al. (1985) FEBS Lett. 190, 37-40]. The base dynamics can be detected in one-dimensional ¹H NMR spectra as excess line width of the aromatic proton resonances. The magnitude of the excess line width is temperature dependent and reaches a maximum at some temperature. In order to better understand the origin of the dynamics, we have studied the effect of N6-methylation of the TpA adenine on both the line widths and its local structure. Here, solution-state 500 and 750 MHz ¹H NMR data collected on [d(CGAGGTTTAAACCTCG)]2 show that the excess line width of theTpA adenine-H2 is diminished when the TpA adenine is N6-methylated and that the line width no longer experiences a maximum as the temperature is varied. The resonances sharpen upon methylation because the amplitude of base motion is restricted due to steric effects and due to other structural changes at the TpA site. Additionally, both the TpA adenine-H8 and the exchangeable imino resonance of thymine at the TpA step were also found to have excess line width that is diminished upon N6-methylation. In order to elucidate the structural features responsible for TpA base dynamics, solution-state NMR structures of [d(CGAGGTTTAAACCTCG)]2 and its A9 N6-methylated derivative were determined at 750 MHz. Comparison of the structures shows that poor base stacking at the TpA step may contribute to, or be the origin of, its base dynamics.

The widely used antitumor drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) reacts with DNA, cross-linking two purine residues through the N7 atoms, which reside in the major groove in B-form DNA. The solution structure of the short duplex [d(CATAGCTATG)]2 cross-linked at the GC:GC site was determined by nuclear magnetic resonance (NMR). The deoxyguanosine-bridging cis-diammineplatinum(II) lies in the minor groove, and the complementary deoxycytidines are extrahelical. The double helix is locally reversed to a left-handed form, and the helix is unwound and bent toward the minor groove. These findings were independently confirmed by results from a phase-sensitive gel electrophoresis bending assay. The NMR structure differs markedly from previously proposed models but accounts for the chemical reactivity, the unwinding, and the bending of cis-DDP interstrand cross-linked DNA and may be important in the formation and repair of these cross-links in chromatin.

A new homonuclear dipolar recoupling technique is described which uses a sequence of phase-shifted, windowless irradiations applied synchronously with sample spinning. Experiments performed on a series of doubly labeled dicarboxylic acids, alanine-1,3-¹³C2, and 2′-deoxythymidine-4,6-¹³C2 demonstrate that this new windowless dipolar recoupling pulse sequence can accurately determine internuclear distances from polycrystalline solids in cases where the coupled spins have large chemical shift anisotropies and large differences in isotropic chemical shift.

Deuterium decoupled proton spectra of the chain region of a partially deuteriated liquid crystal have been taken. The liquid crystal is a trimethylene chain with perdeuteriated cyanobiphenyloxy groups on either end: 1,3-bis(4,4[script ‘]-cyanobiphenyloxy-d8) propane (BCBO3-d16). The spectra have been analyzed to give the dipolar couplings within the propyl chain at various temperatures in the nematic phase of the liquid crystal. These couplings are the most sensitive measurements of molecular flexibility that have yet been taken of an alkyl chain in a pure liquid crystal molecule and give a unique insight into rotational potentials of the flexible component essential to a liquid crystal molecule. The couplings have been analyzed using a maximum entropy method designed for a two-rotor system; in our case the two C–C bonds in the propyl chain. The analysis was also performed by adding to the data set the deuterium quadrupolar splittings of the chain hydrogen atoms, previously measured on the same molecule but deuteriated in the chain. The method picks out the flattest distribution consistent with the data. This also happens to be the most likely distribution given only the data used in the analysis and no other constraints.The results for BCBO3-d16 show a probability maximum for a partially eclipsed conformation (twisted approximately 86° in opposite directions about each C–C bond from the all-trans conformation), a smaller maximum for the all-trans conformation itself, and no significant probability for any of the gauche conformations. The possible motions, that are suggested by these results, therefore, are concerted librations (±86°) between the all-trans position and the partially eclipsed conformations, and full (360°) rotations. These results are in stark contrast to the assumptions made in the rotational isomeric state (RIS) model that the probability distribution can be well approximated by constraining the molecules to exist only in trans and/or gauche states with fast isomerization between such states. In fact the analysis rules out a rotational isomeric state distribution altogether, since our data could not be reproduced by restricting the distribution in this way. Instead it is shown that other distributions are not only possible, but also more likely than any kind of constrained trans–gauche model. ©1994 American Institute of Physics.

A general treatment of spin dynamics during coherence transfer is presented as an example of a many-body problem under strong coupling. In a previous paper, coherence transfer by isotropic mixing (IM) between two coupled spins was examined. However, the greatest experimental interest in the technique of IM centers on the transfer of coherence between spins that are not directly coupled. The dynamics of coherence transfer in multiple spin systems are cast in a superoperator formulation, in which diagonalization of the mixing Liouvilian matrix allows derivation of the coherence transfer frequencies from the eigenvalues. Development of a particular basis of the spin density operator space facilitates diagonalization of the strong coupling Liouvillian. This basis is completely reduced with respect to rotation, permutation, and particle number. Treatment of the many-body aspect is based on an approach using the Young tableaux. The exact calculation of the dynamics of coherence transfer in a three spin system is accomplished using the tableaux method, and closed form solutions are compared with results obtained using numerical methods. The degree to which practical coherence transfer pulse sequences deviate from ideal isotropic mixing, and the effect that these deviations have on symmetry-induced selection rules is calculated numerically.

This article describes systems in which the precession of a single particle spin is magnetically coupled to the excitation of an oscillator. The behavior of such systems resembles that of a “folded” Stern–Gerlach experiment, in which the linear spatial trajectory of the original Stern–Gerlach experiment is folded into the cyclic trajectory of an oscillator. Both quantum and semiclassical solutions to the equations of motion are derived. The results encompass any kind of oscillator which couples to a magnetic field. Examples include mechanical cantilevers with a magnetic source affixed to them, as well as inductor-capacitor resonant circuits. One potential application of oscillator-coupled magnetic resonance is the imaging of biological molecules. Some design issues relevant to molecular imaging are discussed. Review of Scientific Instruments is copyrighted by The American Institute of Physics.

A controlled coherence-transfer experiment is demonstrated experimentally which uses several chemical-shift selective tailored TOCSY mixing periods. The multiple-step tailored TOCSY experiment can dramatically increase the sensitivity relative to a boardband TOCSY experiment and offers the possibility of experimental recognition of specific coupling patterns.

We introduce a new class of semi-windowless pulse sequences for homonuclear decoupling in solids. The development and optimization of the multiple-pulse sequences relies on the definition of quality factors that reflect the proximity of the created effective Hamiltonian to the desired Hamiltonian. A quality factor based on the average Hamiltonian theory gives a global view of the existing solutions. However, the sequences were optimized using a quality factor based on exact numerical solutions of the Liouville von Neumann equation. The new decoupling sequences are stable over a wide range of coupling constants and chemical shifts. The Journal of Chemical Physics is copyrighted by The American Institute of Physics.

Asymmetric amplitude modulated radio frequency pulses for narrow-band inversion of coupled and uncoupled spin systems in NMR are introduced. We also introduce a new parameterization for pulse shapes using cubic spline interpolation that reduces the number of parameters. The spin response is obtained using a full density matrix calculation and pulse shapes were optimized numerically using a simplex algorithm. Coupling is shown to interfere with the performance of shaped pulses designed to invert uncoupled spins prompting the design of pulses that invert a model system consisting of two dipolar coupled spins 1/2. Amplitude modulated shaped pulses optimized for coupled systems are found to be specific for coupling strength and spin topology.

Deuterium NMR of the [methyl ²H ]-2[script ‘]-deoxythymidine labeled synthetic oligonucleotide, [d(CGCGAAT*T*CGCG)]2 in a liquid crystal phase are reported. This ordered phase was observed for hydrated samples with DNA concentrations ranging from 490 to 722 mg ml–1. Temperature variation of line shape and quadrupolar echo decay times were investigated, allowing the degree of alignment and changes in dynamics to be determined. At greatly reduced temperatures a cylindrical line shape was observed, as is expected for samples with the DNA helix axis aligned perpendicular to the magnetic field, while at ambient temperatures, motion about the helix axis averages this cylindrical line shape. NMR investigations at magnetic field strengths of 11.7, 9.4, and 4.7 T resulted in only minor variation in the degree of alignment.

Computer simulations of coherence transfer functions under the isotropic mixing hamiltonian are presented for amino acids and the deoxyribose moiety in DNA. They allow the determination of the optimum mixing time in a two-dimensional TOCSY experiment. The effects of experimental parameters and pulse imperfections are discussed for practical pulse sequences.

Multiple pulse sequences that are tailored to transfer coherence only between coupled spins located in selected regions of chemical shifts are introduced. These sequences can reduce the effective size of complicated coupling networks which results in simplified coherence transfer functions and offers the possibility of spin pattern selective coherence transfer.

Homonuclear coherence transfer experiments, exemplified by the so-called COSY technique, make up an important class of two-dimensional N.M.R. methods. The COSY experiment is useful because coherence transfers in weakly coupled spin systems are confined to spin pairs that are directly coupled, and this restriction may be exploited as a sequential assignment technique. In practice, sequential assignments by COSY methods alone may be very difficult especially if the spin system is complicated (e.g., biopolymers) and the cross peaks in the corresponding COSY spectrum are poorly resolved. Spectral assignments often may be made by relayed coherence transfers, however, where coherence is transferred between remote spins through an intervening network of weakly coupled spins. Recently, a series of experiments have been proposed to effect coherence transfers between remote spins through the application of trains of pulses during the mixing period of a two-dimensional experiment. In this paper we propose a theory of these isotropic mixing methods based in the superoperator formalism, and treat the effect of pulse imperfections on such experiments for the two-spin case. In a subsequent manuscript a generalization of this theory based upon the Young tableaux formalism is applied to larger spin systems. The Young tableaux have been applied to strong coupling problems in atomic and nuclear spectroscopy, and enable a decomposition of the state space and coherent state space of a multiple-particle system into simultaneous irreducible representations of the rotation and permutation groups.

Experimental results demonstrate that recently introduced COMARO decoupling sequences can be used for broadband heteronuclear decoupling in solids, providing decoupling performance that is considerably less sensitive to off-resonance effects than cw decoupling and allowing good decoupling with relatively low rf power.

This study demonstrates the mutual orientation of three tensor interactions in a single NMR experiment. The orientation of the ¹⁵N chemical shift tensor relative to the molecular frame has thus been determined in polycrystalline L-[1-¹³C] alanyl-L-[¹⁵N] alanine. The ¹³C–¹⁵N and ¹⁵N–¹H dipole interactions are determined using the ¹H dipole-modulated, ¹³C dipole-coupled ¹⁵N spectrum obtained as a transform of the data in t2. From simulations of the experimental spectra, two sets of polar angles have been determined relating the ¹³C–¹⁵N and ¹⁵N–¹H dipoles to the ¹⁵N chemical shift tensor. The values determined are betaCN =106°, alphaCN =5° and betaNH =–19°, alphaNH =12°. The experiment verifies, without reference to single crystal data, that sigma33 lies in the peptide plane and sigma22 is nearly perpendicular to the plane. The Journal of Chemical Physics is copyrighted by The American Institute of Physics.

The use of time reversal pulse sequences to obtain multiple quantum spectra of molecules in thermotropic liquid crystalline phases is described. Several studies have already demonstrated that in order to obtain multiple quantum coherence of high order in polycrystalline solids, it is necessary to utilize pulse trains that produce a preparation propagator that is the adjoint of the mixing propagator. Such pulse sequences produce multiple quantum powder spectra that are pure absortive, thus avoiding destructive phase interference that would occur if standard multiple quantum pulse sequences were used. However even in cases where all single quantum transitions are well-resolved, standard multiple quantum pulse sequences yield multiple quantum spectra of low signal-to-noise because single quantum coherent states are projected out of phase. Sensitivity may be improved by projecting the full two dimensional transform, but this may not be practical in cases involving moderately large numbers of strongly coupled spin one-half nuclei. If the time reversal sequences are used however single quantum coherent states are projected in phase and the full two dimensional transform need not be calculated. The pure absorption double quantum spectrum of oriented benzene has been obtained using time reversal pulse trains and demonstrates a considerable increase in sensitivity over standard methods. Practical aspects of applying multiple pulse sequences to thermotropic systems are considered.

The proton multiple quantum NMR (MQ NMR) spectrum of methyl-deuterated hexane-d6 is obtained in a nematic solvent. Rough simulation of the six- and seven-quantum spectra is achieved with a simple model of conformer probabilities and ordering. The advantages and prospects of MQ NMR for the conformational analysis of ordered, non-rigid molecules are discussed.

The conformation and internal dynamics of supercoiled pUC 8 DNA (2717 bp) are examined by dynamic light scattering, and the magnitude and uniformity of its torsional rigidity are determined using time-resolved fluorescence polarization anisotropy of intercalated ethidium dye. Neither measurement gives any indication of an appreciably reduced bending or twisting rigidity, or anomalously rapid internal motions. For ³¹P, in supercoiled pUC 8, we measure T2 = (2.0 ± 0.5) × 10-3 s. This lies within the range of present theoretical estimates obtained using normal rigidities. The proton linewidths observed for pUC 8 and pBR322 (4363 bp) DNAs are within a factor of 2-3 of those similarly estimated assuming ordinary rigidities. According to Bendel, Laub and James [(1982) J. Am. Chem. Soc. 104, 6748-6754], supercoiled pIns36 DNA (7200 bp) exhibits an astonishingly long T2 = 1.17 s for ³¹P, a slowest rotational relaxation time, = 5 × 10-9 s, and an enormously reduced bending rigidity. Serious questions raised by these findings are examined here. The 5 × 10-9 s slowest rotational relaxation time is shown to be physically inadmissible. The nmr relaxation theory developed previously by Allison, Shibata, Wilcoxon, and Schurr [(1982) Biopolymers 21, 729-762], is modified to incorporate new results for deformable filaments, which directly introduce the highly nonexponential tumbling correlation function for reorientation of the local helix axis. Essential requirements for a complete calculation of R2, including estimation of the tumbling correlation function and evaluation of the still unknown DIP/CSA cross-term, are described in detail. Slow coil-deformation modes analogous to the Rouse-Zimm modes of linear DNAs are shown to make an important, if not dominant, contribution to the R2 relaxation rate. Geometrical parameters in the theory are chosen to provide good agreement with literature data for 600-bp linear DNA. Using this theory and an informed guess for the tumbling correlation function, we find that the ³¹P-nmr relaxation data of Bendel et al., if correct, necessarily impose on their DNA one or more extreme properties, such as enormously reduced bending or twisting rigidities. In contrast, the same theory yields reasonable agreement with the T2 reported here for ³¹P in supercoiled pUC 8 DNA when its rigidities are assumed to be quite ordinary.

The use of two-dimensional transform techniques in the observation of multiple-quantum transitions in large spin systems in anisotropic environments is described. In the case of partial resolution in the ω2 dimension, it is shown that the signal to noise of the projection onto ω1 of the absolute magnitude two-dimensional multiple-quantum spectrum is considerably greater than that of the Fourier transform of the t2 = 0 cross section. In practical terms, multiple- quantum transitions of very high order may be observed with good signal to noise after acquisition periods much shorter than previously reported by cross section methods.

A general method of assigning the non-exchangeable protons in the nuclear magnetic resonance spectra of small DNA molecules has been developed based upon two-dimensional autocorrelated (COSY) and nuclear Overhauser (NOESY) spectra in 2H₂O solutions. Groups of protons in specific sugars or bases are identified by their scalar couplings (COSY), then connected spatially in a sequential fashion using the Overhauser effect (NOESY). The method appears to be generally applicable to moderate-sized DNA duplexes with structures close to B DNA. The self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been synthesized by the solid-phase phosphite triester technique and studied by this method. Analysis of the COSY spectrum and the NOESY spectrum leads to the unambiguous assignment of all protons in the molecule except the poorly resolved H5′ and H5″ resonances. The observed NOEs indicate qualitatively that, in solution, the d(C-G-C-G-A-A-T-T-C-G-C-G) helix is right-handed and close to the B DNA form with a structure similar to that determined by crystallography

In multiple-quantum n.m.r. spectroscopy of N coupled spins one obtains n-quantum Fourier transform spectra, where n = 0, 1, 2,…, N. The spectra of particular interest are often those with high n, which may be analysable for a complex molecule without the need for isotopic spin labelling. For example, the n = N quantum spectrum is independent of dipolar couplings and gives the total chemical shift. For n = N – 1, one obtains a spectrum analogous to those from all possible singly isotopically labelled molecules (e.g. one doublet corresponding to one singly labelled species for n = 5 in orientated benzene) and for n = N – 2 all possible doubly isotopically labelled species (e.g. three triplets corresponding to three doubly labelled species for n = 4 in orientated benzene). For large n the intensity decreases, so an important question is whether selective excitation of n-quantum transitions is possible, namely, can one design pulse sequences such that only a particular n or set of n’s is excited? This corresponds to the absorption of only groups of n quanta. It is shown that this can indeed be achieved by employing a combination of time-reversal sequences and phase shifts. The principles of the theory are outlined and examples of experimental results for large n in solids and liquid crystals are presented. The discussion includes spectra, statistical treatment of intensities, degree of selectivity and n-quantum relaxation. Selectivity is also possible in detection and examples of the sensitivity enhancement this provides are shown.