Fast helix formation in the b domain of protein a revealed by site-specific infrared probes

• Type of Material: Article
• Authors: Caitlin M. Davis, Emory University; A. Kat Cooper, Emory University; Richard Dyer, Emory University
• Language: English
• Date: 2015-03-10
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2015 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/acs.biochem.5b00037
• Title of Journal or Parent Work: Biochemistry
• ISSN: 0006-2960
• Volume: 54
• Issue: 9
• Start Page: 1758
• End Page: 1766
• Grant/Funding Information: This work was supported by a grant from the National Institutes of Health (R01 GM53640) to R.B.D.
• Keywords: Methionine; Molecular Sequence Data; Protein Folding; Spectrophotometry, Infrared; Protein Structure, Secondary; Staphylococcal Protein A; Molecular Probes; Peptides; Protein Structure, Tertiary; Circular Dichroism; Spectroscopy, Fourier Transform Infrared; Amino Acid Sequence; Kinetics; Molecular Dynamics Simulation; Infrared Rays
• Research Categories: Engineering, Biomedical; Chemistry, Biochemistry; Biology, Microbiology

Abstract

Comparison of experimental and computational protein folding studies can be difficult because of differences in structural resolution. Isotope-edited infrared spectroscopy offers a direct measure of structural changes involved in protein folding at the single-residue level. Here we demonstrate the increased resolution of site-specific infrared probes to the peptide backbone in the B domain of staphylococcal protein A (BdpA). 13C=18O-labeled methionine was incorporated into each of the helices using recombinant protein expression. Laser-induced temperature jumps coupled with infrared spectroscopy were used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation kinetics of the buried helices, solvated helices, and labeled positions were measured independently by probing the corresponding bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe a fast nanosecond phase and slower microsecond phase at each position. We find at least partial formation of helices 1-3 in the fast intermediate state that precedes the transition state. These measurements provide direct, time-resolved experimental evidence of the early formation of partial helical structure in helices 1 and 3, supporting folding models proposed by computer simulations.