Carbon-deuterium bonds as an infrared probe of protein dynamics, local electrostatics and folding

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 presente

Single Plasmonic Nanoparticle Tracking Studies of Solid Supported Bilayers with Ganglioside Lipids

Single-particle tracking experiments were carried out with gold nanoparticle-labeled solid supported lipid bilayers (SLBs) containing increasing concentrations of ganglioside (GM₁). The negatively charged nanoparticles electrostatically associate with a small percentage of positively charged lipids (ethyl phosphatidylcholine) in the bilayers. The samples containing no GM₁ show random diffusion in 92% of the particles examined with a diffusion constant of 4.3(±4.5) × 10^–9 cm²/s. In contrast, samples containing 14% GM₁ showed a mixture of particles displaying both random and confined diffusion, with the majority of particles, 62%, showing confined diffusion. Control experiments support the notion that the nanoparticles are not associating with the GM₁ moieties but instead most likely confined to regions in between the GM₁ clusters. Analysis of the root-mean-squared displacement plots for all of the data reveals decreasing trends in the confined diffusion constant and diameter of the confining region versus increasing GM₁ concentration. In addition, a linearly decreasing trend is observed for the percentage of randomly diffusing particles versus GM₁ concentration, which offers a simple, direct way to measure the percolation threshold for this system, which has not previously been measured. The percolation threshold is found to be 22% GM₁ and the confining diameter at the percolation threshold only ∼50 nm