Transition State Stabilization
From molecular dynamics simulations, our lab has developed many hypotheses about protein folding/unfolding pathways and native state dynamics. Using protein design, these hypotheses can be tested. For example, based on molecular dynamics simulations of cytochrome b5, it was thought that a cleft near the heme cofactor underwent motion on the ns-ps timescale. Experimentally, in collaboration with the Atkins lab, a disulfide bond was engineered in the cleft. This disulfide bond made the cleft more rigid, confirming the original hypothesis.
In addition, based on extensive high temperature unfolding simulations, our lab predicted a transition state ensemble for chymotrypsin inhibitor 2. Based on the predicted transition state ensemble, our lab, in collaboration with the Fersht lab, made mutations expected to stabilize the transition state ensemble. In vitro, these mutations increased the folding rate of the protein by a factor of 40, providing evidence that the predicted transition state ensemble was correct.
Currently, we are using protein design to test the α-sheet hypothesis. The α-sheet hypothesis is that the α-sheet is the toxic conformer in amyloidogenic proteins such as the prion protein, transthyretin, β2-microglobulin, poly-glutamine, human lysozyme D67H, and super oxide dismutase. The goal of this project is to use molecular dynamics simulations to design a short peptide that will adopt an α-sheet structure. Then, we can use this peptide to determine the spectroscopic signature of the α-sheet, and we can look for this signature in proteins undergoing aggregation. If this spectroscopic signature appears during the course of protein aggregation, then we will have experimental evidence for the α-sheet hypothesis.
We have begun evaluating these designs against the model amyloidogenic protein transthyretin. Interaction of our designs with the protein as it is aggregating will show either an increase or decrease in amyloid formation. Several designs already show promising results, and have formed the basis of further rounds of designs. We are also in the process of immobilizing these designs to test their effectiveness as diagnostic agents.
Also, using molecular dynamics, our lab has predicted the structure of the Prion protein protofibril. This structure is known as the spiral model, and we will test this predicted model using protein design. We will pick mutations that are expected to destabilize the spiral model. Then, these mutant proteins will be expressed and the protofibril-forming propensity will be determined. If the model is correct, these mutant prion proteins will be less likely to undergo aggregation.
- Ladurner A.G., Itzhaki L.S., Daggett V., and Fersht A.R. Synergy Between Simulation and Experiment in Describing the Energy Landscape of Protein Folding, Proceedings of the National Academy of Sciences USA 95: 8473-8478, 1998. [PDF] [HTML]
- Storch E.M., Daggett V., and Atkins W.M. Engineering Out Motion: Introduction of a de novo Disulfide Bond and a Salt Bridge Designed to Close a Dynamic Cleft on the Surface of Cytochrome b5, Biochemistry 38: 5054-5064, 1999. [DOI]
- Storch E.M., Grinstead J.S., Campbell A.P., Daggett V., and Atkins W.M. Engineering Out Motion: A Surface Disulfide Bond Alters the Mobility of Trp 22 in Cytochrome b5 as Probed by Time-Resolved Fluorescence and 1H-NMR Experiments, Biochemistry 38: 5065-5075, 1999. [DOI]
- Hom K., Wolfe G., Ma Q.-F., Zhang H., Storch E.M., Daggett V., Basus V.J., Waskell L. NMR Studies of the Association of Cytochrome b5 with Cytochrome c: Biochemistry 39: 14025-14039, 2000. [DOI]