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Solid-state NMR measurements of depths and distances in membrane peptides and proteins

Gregory J Gallagher, University of Massachusetts Amherst


The limitations on the structural studies of membrane proteins prevent the elucidation of the mechanism of transmembrane signaling. A recently developed solid-state NMR technique named spin diffusion allows for depth measurements from the membrane surface to be conducted on membrane peptides or proteins. This technique shows promise for the determination of protein orientation and structure in the membrane. We demonstrate the resolution of spin diffusion on a membrane-bound peptide of known structure, gramicidin A. Depth differences of 5.0Å are measured using three samples each with a single 15N-backbone labeled site. We conclude that spin diffusion can determine the orientation of a membrane-bound peptide and distinguish between structural models. However it is of limited use for high-resolution depth measurements in membrane proteins due to the large (70%) sensitivity loss inherent to the experiment. While the above technique may not be best suited for membrane proteins, REDOR and rotational resonance have provided high-resolution distance measurements on membrane peptides and proteins. The utility of these experiments made both of them the subject for calibration and procedural improvements. We demonstrate a procedure to correct for the 13C natural abundance contribution to the rotational resonance experiment in a membrane protein (the serine receptor of bacterial chemotaxis). We show the accuracy of this procedure by measuring a known 5.0Å distance in a 13CO-Tyr, 13C β-Cys labeled receptor sample. High-resolution distance measurements can be made in native membrane proteins using both REDOR and rotational resonance. We employed 13 C{19F} REDOR to measure two interhelical distances in the transmembrane domain of the serine chemoreceptor from E. coli . These two distances and a 13C-13C distance previously measured by rotational resonance distance show no significant changes upon ligand binding to the receptor. Although some of these distances are significantly longer than predicted by the structural model of the transmembrane domain, the NMR distance constraints can be satisfied by plausible structures (structures retaining alpha helical backbone conformation). Although the 12 models that were generated vary enough to accommodate small conformational changes such as the 1.6Å piston, the NMR data provide high-resolution distance constraints with no evidence for a ligand-induced conformational change in the transmembrane domain. These NMR results support models in which a conformational change is not propagated by the transmembrane helices. Combining this with the growing evidence from other investigators that receptor clusters play a role in signaling, we suggest that ligand binding induces a local conformational change in the periplasmic domain which alters receptor interactions to modulate clustering.

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Recommended Citation

Gallagher, Gregory J, "Solid-state NMR measurements of depths and distances in membrane peptides and proteins" (2006). Doctoral Dissertations Available from Proquest. AAI3206208.