The objective of the proposed research is to demonstrate methods to assign chemical shifts and shift anisotrophies on uniformly labeled membrane proteins in the solid state. This is a challenging project however, no uniformly 13C, 15N labeled protein of even moderate size has yet been fully assigned in the solid state. There have been significant improvements in NMR methodology (pulse sequence, hardware) which point to this as a possibility. In particular, dipolar recoupling techniques with higher efficiencies, advanced decoupling methods, probes capable of 25-30 kHz MAS and 200 kHz decoupling fields, and widebore magnets with 1H frequencies of 750 MHz should lead to full sold-state NMR structure determinations of membrane proteins. The chemical shifts in the retinal binding pocket will be particularly interesting to analyze, given the significant interactions between the sidechains and retinal. These shifts will be used along with quantum chemical methods to analyze in detail the mechanism by which the proton is pumped uniaxially.
| Su, Wei; Slevin, Michael K; Marzluff, William F et al. (2016) Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA. Methods Mol Biol 1428:93-114 |
