"Use of a Paramagnetic Spin Label for Determination of Long-Range Distance Constraints in Solid-State NMR"
Solid-State Nuclear Magnetic Resonance (SSNMR), along with paramagnetic relaxation enhancement (PRE), is used to obtain long-range distance constraints in the protein Proteorhodopsin (PR). PRE allows for the acquisition of long-range distance constraints not accessible through standard NMR experiments, allowing for the determination of a low resolution template protein structure which can be further refined. Four mutant versions of PR, created to have only one cysteine residue present in each, were investigated. Mass spectrometry was used to determine the purity of the protein sample as well as the extent the sample was labelled with a paramagnetic tag. The S215C mutant was found to not purify well and appeared to retain a large amount of detergent. No further studies were carried out with this mutant. The S55C variant was found to differ significantly from the wild-type structure based on chemical shift comparisons. This difference is thought to be due to a lack of oligomeric structure in the lipid bilayer, as electron paramagnetic relaxation studies did not detect the expected distance between spin labels in the reconstituted sample. The C107 mutant experienced delocalized PRE effects that were originally thought to be due to proton spin diffusion, but when this interaction was removed to the first order by the use of Lee-Goldburg cross-polarization (CP), the delocalized effect was still observed. The C175 mutant produced usable long-range distance restraints that can be used to restrain three of the helices in the bundle on the cytoplasmic side. Future studies using PREs with PR are required to better understand the structure of this membrane protein.
Dr. Hermann Eberl, CHAIR (Department of Mathematics & Statistics)
Dr. Leonid Brown, CO-ADVISOR (Department of Physics)
Dr. Vladimir Ladhizansky, CO-ADVISOR (Department of Physics)
Dr. Steffen Graether, EXAMINATION COMMITTEE MEMBER (Dept. of Molecular and Cellular Biology)