期刊
CHEMPHYSCHEM
卷 15, 期 5, 页码 849-853出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cphc.201400017
关键词
labeling; IR spectroscopy; perturbation; protein environments; protein folding
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 (CD), cyano groups (CN), and azides (N-3); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N-terminal Src homology3 domain of the murine adapter protein Crk-II (nSH3) with CD 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 N-3 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 CD labels, which are both sensitive to their environment and non-perturbative, CN and N-3 probes should be used with caution.
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