Characterizing the Structure of Surface-Immobilized Proteins: A Surface Analysis Approach

Citation

Joe, E. Baio; Tobias, Weidner; & David, G. Castner (2012). Characterizing the Structure of Surface-Immobilized Proteins: A Surface Analysis Approach. In Proteins at Interfaces III State of the Art (pp. 761-779). American Chemical Society.

Abstract

There are many techniques that allow surface scientists to study interfaces. However, few are routinely applied to probe biological surfaces. The work presented here demonstrates how detailed information about the conformation, orientation, chemical state, and molecular structure of biological molecules immobilized onto a surface can be assessed by electron spectroscopy, mass spectrometry, and nonlinear vibrational spectroscopy techniques. This investigation began with the development of simple model systems (small proteins, and peptides) and has evolved into a study of more complex ? real world systems. Two model systems based on the chemical and electrostatic immobilization of a small rigid protein (Protein G B1 domain, 6kDa) were built to develop the capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS), near edge X-ray absorption fine structure spectroscopy (NEXAFS) and sum frequency generation (SFG) spectroscopy as tools to probe the structure of surface immobilized proteins. ToF-SIMS sampled the amino acid composition of the exposed surface of the protein film. Within the ToF-SIMS spectra, an enrichment of secondary ions from amino acids located at opposite ends of the proteins were used to describe protein orientation. SFG spectral peaks characteristic of ordered α-helix and ?-sheet elements were observed for both systems and the phase of the peaks indicated a predominantly upright orientation for both the covalent and electrostatic configurations. Polarization dependence of the NEXAFS signal from the N 1s to π* transition of the peptide bonds that make up the ?-sheets also indicated protein ordering at the surface.

Reference Type

Book Section

Secondary Title

Proteins at Interfaces III State of the Art

Author(s)

Joe, E. Baio
Tobias, Weidner
David, G. Castner

Year Published

2012

Date Published

1325376000

Volume Number

1120

Issue Number

1120

Pages

761-779

Publisher

American Chemical Society

DOI

Characterizing the Structure of Surface-Immobilized Proteins