R. Adhikary, J. Zimmermann, F.E. Romesberg, Chem. Rev. (2017) 117:1927–1969.
We review the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study. These topics are put into context through four case studies focused on KSI, SH3, DHFR, and cyt c.
R. Adhikary, J. Zimmermann, P.E. Dawson, F.E. Romesberg, Anal. Chem. (2015) 87:11561–11567.
In both model systems and the N-terminal Src homology 3 domain of Crk-II (nSH3), we show that the absorption frequency of cyano and thiocyano probes is linearly correlated with temperature and that the slope of the resulting line (frequency-temperature line slope or FTLS) reflects the nature of the probe’s microenvironment, including whether or not the probe is engaged in H-bonds.
R. Adhikary, J. Zimmermann, J. Liu, R. Forrest, T. Janicki, P. Dawson, S. Corcelli, F.E. Romesberg, J. Am. Chem. Soc. (2014) 136:13474-13477.
We characterize the IR stretching frequencies of deuterated variants of proline and four proline residues of an Src homology 3 domain protein. CD stretching frequencies are shifted to lower energies due to hyperconjugation with Ni electron density, and along with DFT calculations, the data reveal that the Ni+1-H–Ni interactions constitute H-bonds that may play an important role in protein folding, structure, and function.
R. Adhikary, J. Zimmermann, P.E. Dawson, F.E. Romesberg, ChemPhysChem (2014) 15:849-853.
We use IR spectroscopy to characterize nSH3 variants labeled with CN or N3 at five different positions. Like carbon-deuterium (C-D) bonds, CN and N3 can provide information about their surrounding protein environment, but unlike C-D, they tend to be perturbative and thus should be used with caution.
J. Zimmermann, F.E. Romesberg, Methods Mol. Biol. (2014) 1084:101-19.
We describe the experimental procedures required to use C-D bonds and FT IR spectroscopy to characterize protein dynamics, structural and electrostatic heterogeneity, ligand binding, and folding.
R. Adhikary, J. Zimmermann, J. Liu, P.E. Dawson, F.E. Romesberg, J. Phys. Chem. B, 117:13082-13089.
We use the IR absorptions of carbon-deuterium bonds site-selectively incorporated throughout the N-terminal SH3 domain from the murine adapter protein CrkII to characterize its different microenvironments with high spatial and temporal resolution.
W. Yu, P. Dawson, J. Zimmermann, F.E. Romesberg, J. Phys. Chem. B (2012) 116:6397-6403.
We report a residue-specific characterization of the thermal unfolding mechanism of cyt c using C-D bonds site-specifically incorporated at residues dispersed throughout three different structural elements within the protein. Elucidation of the detailed unfolding mechanism and the structure of the associated molten globule, both of which represent challenges to conventional techniques, are highlights of the utility of the C-D technique.