Carbon-deuterium bonds as an infrared probe of protein dynamics, local electrostatics and folding
- Author(s): Sagle, Laura B.;
- et al.
The new technique being developed in the Romesberg laboratory of incorporating carbon-deuterium bonds into proteins and using them as infrared probes is further explored. Carbon-deuterium bonds are incorporated into horse heart cytochrome c through its semi-synthesis in which only the C-terminal 39 residues are accessible. Chapter 3 describes a project investigating redox-liked differences in cytochrome c by the incorporation of C-D bonds at six residues throughout the protein. It is found that when the protein is oxidized, there are both electrostatic changes as well as a greater amount of unfolded protein present only on the proximal side of the heme. The lack of consistent linewidth changes, indicating greater flexibility of the protein in the oxidized state, along with distinct changes in the amount of unfolded protein present suggests an alternative explanation for the difference in the two redox states of cytochrome c. The data indicates that there is in fact no difference in flexibility between the reduced and oxidized states of the protein, but rather a change in the unfolding equilibrium, giving rise to more unfolded protein in the oxidized state. Subsequent chapters describe the development of using C-D bonds as infrared probes for protein folding. Six residues throughout the protein were characterized as cytochrome c was unfolded in both GnHCl and Urea. In GnHCl, the unfolding of the protein is cooperative with the exception of the Met80 loop, which undergoes an intermediate most likely due to misligation. In urea, the unfolding mechanism is quite different, and a sequential unfolding pathway similar to that observed in the amide- exchange NMR studies is presented. Along with a sequential unfolding pathway, some new observations on the folding of cytochrome c in urea have resulted. Although a similar misligated intermediate involving the Met80 loop is observed in urea, some notable and interesting differences from the GnHCl data are discussed. The possibility of a new cooperative folding unit containing the Met80 loop and the 60's helix also presents itself when the protein is unfolded in urea. Lastly, the high resolution data revealing the sequential unwinding of the C-terminal helix from the C-terminus is presented