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.
J. Zimmermann, M.C. Thielges, Y.J. Seo, P.E. Dawson, F.E. Romesberg, Angew. Chem. Int. Ed. (2011) 50:8333-8337.
The cyano group is sensitive to its environment, absorbs in a unique region of the protein IR spectrum, and may be appended to an amino acid. Using both steady-state and time-resolved methods, we explore the use of cyano groups as probes of protein microenvironments and dynamics in variants of cytochrome c. We find that the cyano group is a useful site-specific probe of protein microenvironments and dynamics, but that it can also perturb its environment and destabilize the folded state of the protein.
J. Zimmermann, M.C. Thielges, W. Yu, P.E. Dawson, F.E. Romesberg, J. Phys. Chem. Lett. (2011) 2:412-416.
We explore the use of carbon-deuterium (C-D) bonds under time-resolved conditions to follow the unfolding of cytochrome c from a photostationary state that accumulates after CO is photodissociated from the protein’s heme prosthetic group. Our results clearly show that C-D bonds are well-suited to characterize protein folding and dynamics.
P. Weinkam, J. Zimmermann, F.E. Romesberg, P. Wolynes, Acc. Chem. Res. (2010) 43:652-660.
At low pH, the experimentally observed folding sequence of cyt c deviates from that at pH 7 and from models with perfectly funneled energy landscapes. We account for these alternative folding pathways using complex models that begin from native structure-based terms and also add the chemical frustration that arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH.
M.T. Thielges, J. Zimmermann, F.E. Romesberg, J. Am. Chem. Soc. (2009) 131:6054-6055.
WeWe address the processes that occur upon folding of reduced cyt c induced by photodissociation of CO from the CO-bound, unfolded protein by monitoring the CO vibration with step-scan FT-IR spectroscopy.
M.C. Thielges, J. Zimmermann, P.E. Dawson, F.E. Romesberg, J. Mol. Biol. (2009) 24:159-167.
Using site-selectively incorporated carbon–deuterium bonds, we show that like equine cyt c, bovine cyt c is induced to unfold by guanidine hydrochloride via a stepwise mechanism, but it does not populate an intermediate as is observed with the equine protein.
P. Weinkam, F.E. Romesberg, P.G. Wolynes, Biochemistry (2009) 48:2394-2402.
A grand canonical formalism is developed to combine discrete simulations for chemically distinct species in equilibrium. Each simulation is based on a perturbed funneled landscape. The formalism is illustrated using the alkaline-induced transitions of cyt c as observed by FT-IR spectroscopy and with various other experimental approaches.