Abstract
Bacteriorhodopsin (bR) is a 26 kDa archaeal membrane protein that harvests light energy to create an ion gradient across the cell membrane. Photoisomierzation of the retinilydene chromophore is coupled to ion translocation via a sequence of photocycle intermediates. Here we apply selective multidimensional solid-state NMR to uniformly 13C,15N-labeled bR in its native membrane to obtain chemical shifts in the chromophore of cryogenically trapped bR photointermediates. This is made feasible by using 250 GHz radiation to stimulate dynamic nuclear polarization (DNP), whereby the large spin polarization of unpaired electrons in exogenous free biradicals is transferred to nuclei. Subsequent N-C-C transfers in the NMR experiment allow us to distinguish four discrete substates of the L intermediate. Three of these are shunts that revert to the resting state of the protein upon thermal relaxation, while one L substate, labeled as ‘persistent L’ in our earlier 1D experiments, relaxes to the M state and is therefore deemed functional. Functional L has the strongest counterion, as indicated by its Schiff base (SB) nitrogen chemical shift. It also has a fully planarized 13-cis C13=C14 bond, as indicated by the gamma effect on the C12 chemical shift. These results are consistent with indications from time-resolved optical spectrometry and QM/MM studies of multiple barriers on the way to SB deprotonation. On the other hand, they are inconsistent with models in which the C13=C14 bond is twisted until Schiff base deprotonation. The experiments also demonstrate the use of DNP-enhancement at cryogenic temperatures to investigate mixed states of a membrane protein by multi-dimensional NMR. The results presented here would have been impossible without the availability of DNP to enhance spin polarization that is spread over multiple atoms in multiple protein states.