Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

Abstract

Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Here, we demonstrate that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes. In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site.

MAS NMR detection of hydrogen bonds for protein secondary structure characterization

“…While transfers to directly bonded nuclei are most efficient, CP is a dipolar process, and thus also useful for detecting through-space interactions in the sparsely protonated context of a back-exchanged sample. Accordingly, it has been shown earlier that protons in hydrogen bonds and hydroxyl protons can be detected by solid-state MAS NMR using CP (Agarwal et al 2010(Agarwal et al , 2013Friedrich et al 2020).…”

Directional Proton Conductance in Bacteriorhodopsin Is Driven by Concentration Gradient, Not Affinity Gradient

“…Since several water molecules have been observed in the inner EC channel, possibilities of other proton complexes also exist, such as H 5 O 2 + (Marx et al, 1999; Mathias and Marx, 2007). The hydronium 402 has been shown possible by magic angle spinning NMR (Friedrich et al, 2020).…”

“…The inner EC channel has been called a proton cage at the active site of bR (Friedrich et al, 2020), which reflects the same fact of the proton barrier (Fig. 5).…”

Protein structural dynamics by Magic-Angle Spinning NMR

“…Similarly, the protonation dynamics of the active site of bacteriorhodopsin was monitored by 1 H chemical shift exchange experiments and revealed slow oscillatory motions of protons in the dark state. Such protonation dynamics is thought to be disrupted in the light-activated states, thus promoting the functional proton pumping [48] .…”