Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces
We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.
KeywordsDynamic nuclear polarization DNP Membrane proteins Solid-state NMR Gramicidin DEER spectroscopy
This work was supported by grants from the National Institutes of Health and National Science Foundation. Professor McDermott is a member of the New York Structural Biology Center. The Center is a STAR center supported by the New York State Office of Science, Technology, and Academic Research. NMR resources were supported by NIH P41 GM66354. This work was supported by NSF MCB 0749381, NIH R01 GM 88724 to A. E. M., NIH NRSA F32 087908 to B. J. W. and by NIH/NIGMS P41GM103521 and NIH/NIBIB R01EB003150 to J.H.F.
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