C15=N Torsion Measured by DNP-Enhanced Solid State NMR in Bacteriorhodopsin Intermediates

Abstract

Bacteriorhodopsin is a 26 kDa light-driven ion pump that establishes an ion gradient across the membrane of Halobacterium salinarium. Although it has been characterized extensively by a wide range of techniques, structural details pertinent to its mechanism are still under scrutiny. Of particular interest is chromophore torsion that would orient the protonated Schiff base favorably toward the proton acceptor until proton transfer occurs. Thus, a measurement of the C15=N torsion in L, the intermediate directly preceeding proton transfer, would yield evidence supporting one of the models proposed for the proton transfer. By performing dipolar recoupling between 13C labels at retinal-C14 and Lys-Ce, we determined the distance between the labeled sites, and thus the torsion angle around C15=N.Utilizing the sensitivity available with DNP (Dynamic Nuclear Polarization), only 7.4 hours is needed to record a 2D spectrum from 15 mg of protein, even when the intensities of interest are divided in a roughly 60:40 ratio, corresponding to two different intermediates. This demonstrates the utility of DNP-SSNMR in obtaining precise quantitative measurements in membrane proteins, even in mixed states.Figure 1SSNMR recoupling build-up curve of retinal-14C, K216-Ce distance in bacteriorhodopsin. Data were fit to yield 3.11 +/− 0.02A between 14C and Ce in bR555, and 3.90 +/−0.08 A in bR568.View Large Image | View Hi-Res Image | Download PowerPoint Slide