title: IR probes of protein microenvironments: utility and potential for perturbation.
abstract: A variety of IR-active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon-deuterium bonds (CD), cyano groups (CN), and azides (N3 ); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N-terminal Src homology 3 domain of the murine adapter protein Crk-II (nSH3) with CD bonds site-selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two-state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N3 probes at five of the same positions. In agreement with previous studies, the position-dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface-exposed positions. Thus, unlike CD labels, which are both sensitive to their environment and non-perturbative, CN and N3 probes should be used with caution.
authors: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA).
authors: Ramkrishna
authors: R
authors: Adhikary
authors: Jörg
authors: J
authors: Zimmermann
authors: Philip E
authors: PE
authors: Dawson
authors: Floyd E
authors: FE
authors: Romesberg
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry*; D017510:Protein Folding; D050737:Proto-Oncogene Proteins c-crk / Q000737:chemistry* / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013696:Temperature
chemical_list: D001386:Azides; D003486:Cyanides; D015335:Molecular Probes; D050737:Proto-Oncogene Proteins c-crk; D003903:Deuterium
keywords: IR spectroscopy; labeling; perturbation; protein environments; protein folding