pH-sensitive vibrational probe reveals a cytoplasmic protonated cluster in bacteriorhodopsin

Abstract

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin.

Allosteric Effects of the Proton Donor on the Microbial Proton Pump Proteorhodopsin

“…(27). More recently, it was revealed that proton uptake in bR utilizes a cluster of residues to stabilize interactions of the proton donor with bulk cytoplasmic waters, leading to modulation of the later stages in the photocycle (28). PR does not possess a corresponding aspartic acid residue, so it would make sense that the proton donor behaves differently from bR.…”

“…This rapid release of the helical bundle in bR (i.e., ''latch mechanism'') has a twofold purpose: 1) to form the water wire and 2) to prevent backflow of protons that impede the photocycle (27). Although PR has a glutamic acid (E108) in the same position as the proton donor in bR, it lacks a pair of glutamic acids that act as a proton uptake complex (28) as well as a hydrogen-bonding partner in helix B that lies directly across from the proton donor (12). In addition, an acidic residue as the proton donor is not conserved among other PR-like proton pumps, such as a lysine in Elasmotherium sibiricum rhodopsin (29) and a histidine on helix B in eubacterial proton pumps (30).…”

Light‐Gated Synthetic Protocells for Plasmon‐Enhanced Chemiosmotic Gradient Generation and ATP Synthesis

“…Bacteriorhodopsin in AuAgNR SPCs showed the highest proton transporting rate, which was about 1.9‐ and 2.7‐fold higher than those of AuNP and SiO 2 NP SPCs, respectively. Given the negligible difference in cargo release kinetics of the three CCs studied above (Figures S11–13) and the well‐known accelerated migration of protons assisted by mobile buffer molecules, the possibility that the proton diffusion was the rate‐limiting factor could be excluded. The ability of plasmonic nanostructures to strongly localize incident light close to their surface has been shown to greatly enhance the optical properties of proximal (bio‐)molecules, which generally requires strong spectral overlap between the plasmonic resonance and (bio‐)molecule absorption/emission band .…”

Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies

“…Since its discovery in 1971 [ 1 ], the light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum (previously known as Halobacterium halobium ) has become an intensively studied model membrane protein for proton translocation across biological membranes [ 2 , 3 , 4 , 5 ], photochemical events in proteins [ 6 , 7 , 8 , 9 ], photophosphorylation [ 10 , 11 , 12 ] and structural studies [ 13 , 14 , 15 , 16 ]. The use of BR to study proton transport across membranes in different time scales using time-resolved serial femtosecond crystallography at X-ray free electron lasers [ 17 , 18 , 19 , 20 ] and as an alignment tool for NMR studies [ 21 ] highlights its significant contribution to emerging technologies.…”