Elucidating the pumping mechanism of bacteriorhodopsin using dynamic nuclear polarization enhanced magic angle spinning NMR

Abstract

Bacteriorhodopsin (bR) is comprised of 7 trans-membrane helices that enclose a retinylidene chromophore formed by a Schiff base (SB) between the retinal and Lys216. Due to bR's relative availability, it serves as a model for other members of the rhodopsin family, ion channels and GPCRs. Since its discovery in the 1970's, bR has been intensely studied by various methods including: X-ray crystallography, EM, FT-IR, molecular simulations, and NMR, etc. Despite numerous advances, details of its pump mechanism remain elusive due to experimental limitations in sensitivity and/or resolution. Here, dynamic nuclear polarization (DNP) is employed to boost the ¹H NMR signal. With an enhancement of 75, multidimensional spectra of low gyromagnetic nuclei were made possible. The cryogenic experimental temperature also traps the various bR photocycle intermediates, allowing them to be studied in situ. We are able to answer the one lingering question regarding bR's primary proton transfer pathway and conduct distance measurements near the active site. The pathway of bR's primary proton transfer has been the subject of scrutiny for many years. DNP MAS NMR bond length measurements of the SB proton reveal an elongated N-H bond in L, the transfer of ¹H in deprotonated MO, and a tight N-H bond in N intermediate. The ¹H chemical shift of ~3.6 ppm in M₀ indicates an alcohol hydrogen donor partner. This strongly supports the SB H+ being relayed from the SB to Asp85 via Thr89 as the pathway for bR's primary proton transfer. Distance measurements obtained here are the first set of long-range DNP MAS NMR measurements conducted on a uniformly labeled bR system. We find that the SB-D85 distance shrinks in the first half of the photocycle and is released after the primary proton transfer. The decrease in distance between the two indicates helix C and helix G are moving toward each other, which could be the reason why functional L is difficult to achieve. The subsequent release of helix G provides an additional gate to the release of the torsion energy in the chromophore. Meanwhile, the SB-D212 distance hardly changes.