Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

Abstract

To achieve efficient proton pumping in the light‐driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark‐adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side‐chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground‐state bR.

Side‐chain dynamics of the α1B‐adrenergic receptor determined by NMR via methyl relaxation

“…The obtained methyl order parameters for α 1B ‐AR‐B1D1 bound to the three different inverse agonists cover almost the entire range of possible values (Figure 2b–e ). Interestingly and in contrast to α 1B ‐AR‐B1D1, the other membrane proteins investigated so far had no side chains belonging to the most rigid motional class ω (Kooijman et al, 2020b ; O'Brien et al, 2020 ). The average of the methyl order parameters for α 1B ‐AR‐B1D1 in micelles was 0.51 (Figure 2f ), which is larger than the values reported for bR in nanodiscs (Kooijman et al, 2020b ; mean S 2 axis = 0.41) and for pSRII (O'Brien et al, 2020 ) in micelles (mean S 2 axis = 0.37) or bicelles (mean S 2 axis = 0.45).…”

“…Interestingly and in contrast to α 1B ‐AR‐B1D1, the other membrane proteins investigated so far had no side chains belonging to the most rigid motional class ω (Kooijman et al, 2020b ; O'Brien et al, 2020 ). The average of the methyl order parameters for α 1B ‐AR‐B1D1 in micelles was 0.51 (Figure 2f ), which is larger than the values reported for bR in nanodiscs (Kooijman et al, 2020b ; mean S 2 axis = 0.41) and for pSRII (O'Brien et al, 2020 ) in micelles (mean S 2 axis = 0.37) or bicelles (mean S 2 axis = 0.45). This indicates that α 1B ‐AR‐B1D1 adopts a more rigid structure (on a ps to ns timescale) than the two microbial rhodopsins.…”

“…(d) Average Ile, Leu, and Val methyl order parameters including the 95% confidence interval for the mean. Data for α 1B ‐AR‐B1D1 bound to the indicated ligands and for the published S 2 axis values of bR (Kooijman, Schuster, et al, 2020 ) and pSRII (O'Brien et al, 2020 ) are included. Significances between order parameters were assessed using the Wilcoxon rank‐sum test.…”

“…To shed light on this question, we compared the side‐chain dynamics of α 1B ‐AR‐B1D1 with those of the two microbial rhodopsins bR and pSRII, whose methyl order parameters were published by Kooijman et al ( 2020b ) and O'Brien et al ( 2020 ), respectively (Figure 5d ). α 1B ‐AR, bR, and pSRII share a common architecture of seven TM helices; however, bR is a light‐driven proton pump and pSRII is a photoreceptor for blue light.…”

“…Methyl order parameters have been obtained for several soluble and, so far, three microbial membrane proteins, that is, the β‐barrel outer membrane protein W (OmpW; O'Brien et al, 2020 ), sensory rhodopsin II (pSRII; O'Brien et al, 2020 ), and bacteriorhodopsin (bR; Kooijman et al, 2020b ). Compared with soluble proteins, these three microbial membrane proteins showed increased side‐chain dynamics and the absence of very rigid side chains.…”