Photoinduced Isomerization Sampling of Retinal in Bacteriorhodopsin

Abstract

Photoisomerization of retinoids inside a confined protein pocket represents a critical chemical event in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. Light driven proton pumping in bacteriorhodopsin entails exquisite electronic and conformational reconfigurations during its photocycle. However, it has been a major challenge to delineate transient molecular events preceding and following the photoisomerization of the retinal from noisy electron density maps when varying populations of intermediates coexist and evolve as a function of time. Here I report several distinct early photoproducts deconvoluted from the recently observed mixtures in time-resolved serial crystallography. This deconvolution substantially improves the quality of the electron density maps hence demonstrates that the all-trans retinal undergoes extensive isomerization sampling before it proceeds to the productive 13-cis configuration. Upon light absorption, the chromophore attempts to perform trans-to-cis isomerization at every double bond coupled with the stalled anti-to-syn rotations at multiple single bonds along its polyene chain. Such isomerization sampling pushes all seven transmembrane helices to bend outward, resulting in a transient expansion of the retinal binding pocket, and later, a contraction due to recoiling. These ultrafast responses observed at the atomic resolution support that the productive photoreaction in bacteriorhodopsin is initiated by light-induced charge separation in the prosthetic chromophore yet governed by stereoselectivity of its protein pocket. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable.

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

“…1). Eight intermediate structures along the photocycle, now isolated from one another, are refined against reconstituted structure factor amplitudes (Methods; Table S2) (Ren, 2021). The assignments of the previously identified intermediates I to M to these refined structures are largely based on the time stamps of the observed signals.…”

“…Near perfect 13-cis is successfully refined in both structures of J' at ~700 fs and J at ~20 ps with a contracted retinal binding pocket (Fig. 2b 4 th panel) (Ren, 2021). However, the SB double bond C15=Nz is momentarily distorted from the trans configuration in J' with a torsion angle of 133°.…”

“…An all-trans retinal is covalently connected to Lys216 of helix G through a protonated Schiff base (SB) pointing toward the extracellular side (EC) in the resting state, of which the double bond C15=Nz is also in trans (traditionally also noted as anti (McCarty, 1970)) Upon absorption of a visible photon, the retinal isomerizes efficiently and selectively to adopt the 13-cis configuration (Govindjee et al, 1990). Extensive structural events during the ultrafast isomerization sampling and stereochemical selection were recently revealed by the companion study of this work (Ren, 2021).…”

“…Three decomposed map components U10, U14, and U17 of the short delays contain extraordinary structural signals in terms of their extensiveness and quality (Ren, 2021).…”

“…These signals originate exclusively from a few time points of Nogly et al, too few to fit the time dependency with exponentials. Instead, a spline fitting through these time points estimates the coefficients c10, c14, and c17 in the linear combination of c10U10 + c14U14 + c17U17 for the early states I, J, and their respective precursors I', J' (Ren, 2021). The precursor prior to I state is located on the spline trajectory from the origin, that is, the ground state bR at the time point of 0-before the photon absorption, to the first time point of 49-406 fs (PDB entry 6g7i).…”