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Carbon-deuterium bonds as probes of protein thermal unfolding

W. Yu, P. Dawson, J. Zimmermann, F.E. Romesberg, J. Phys. Chem. B (2012) 116:6397-6403.
pubpic2012yuWe 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.

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Cyano groups as probes of protein microenvironments and dynamics

J. Zimmermann, M.C. Thielges, Y.J. Seo, P.E. Dawson, F.E. Romesberg, Angew. Chem. Int. Ed. (2011) 50:8333-8337.
pubpic2010xxThe 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.

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Carbon-deuterium bonds as site-specific and non-perturbative probes for time-resolved studies of protein dynamics and folding

J. Zimmermann, M.C. Thielges, W. Yu, P.E. Dawson, F.E. Romesberg, J. Phys. Chem. Lett. (2011) 2:412-416.
pubpic2010xxWe 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.

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The folding energy landscape and free energy excitations of cytochrome c

P. Weinkam, J. Zimmermann, F.E. Romesberg, P. Wolynes, Acc. Chem. Res. (2010) 43:652-660.
pubpic2010weinkamAt 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.

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The determinants of stability and folding in evolutionarily diverged cytochromes c

M.C. Thielges, J. Zimmermann, P.E. Dawson, F.E. Romesberg, J. Mol. Biol. (2009) 24:159-167.
pubpic2009thielgesUsing 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.

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Chemical frustration in the protein folding landscape: grand canonical ensemble simulations of cytochrome c

P. Weinkam, F.E. Romesberg, P.G. Wolynes, Biochemistry (2009) 48:2394-2402.
pubpic2009xxA 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.