Experimental Characterization of Electrostatic and Conformational Heterogeneity in an SH3 Domain

• Published in: The journal of physical chemistry. B 2013-10, Vol.117 (42), p.13082-13089
• Main Authors: Adhikary, Ramkrishna, Zimmermann, Jörg, Liu, Jian, Dawson, Philip E, Romesberg, Floyd E
• Format: Article
• Language: English
• Subjects: Adapters; Animals; Biological and medical sciences; Conformational dynamics in molecular biology; Deuterium - chemistry; Electrostatics; Fundamental and applied biological sciences. Psychology; Heterogeneity; Magnetic Resonance Spectroscopy; Mice; Molecular biophysics; Peptides - chemical synthesis; Peptides - chemistry; Physical chemistry; Protein Denaturation; Protein Structure, Tertiary; Proteins; Proto-Oncogene Proteins c-crk - chemical synthesis; Proto-Oncogene Proteins c-crk - chemistry; Spectra; Spectroscopy, Fourier Transform Infrared; src Homology Domains; Static Electricity; Temperature; Temporal resolution; Thermal denaturation; Thermodynamics
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/jp402772x

Electrostatic and conformational heterogeneity make central contributions to protein function, but their experimental characterization requires a combination of spatial and temporal resolution that is challenging to achieve. Src homology 3 (SH3) domains mediate protein–protein interactions, and NMR studies have demonstrated that most possess conformational heterogeneity, which could be critical for their function. Here, we use the IR absorptions of carbon–deuterium (C–D) bonds site-selectively incorporated throughout the N-terminal SH3 domain from the murine adapter protein Crk-II to characterize its different microenvironments with high spatial and temporal resolution. The C–D absorptions are only differentiated in the folded state of the protein where they show evidence of significant environmental heterogeneity. However, the spectra of the folded state are independent of temperature, and upon thermal denaturation the protein undergoes a single, global unfolding transition. While some evidence of conformational heterogeneity is found within the peptide backbone, the majority of the environmental heterogeneity appears to result from electrostatics.