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

Ren, Zhong

doi:10.1101/2021.10.04.463074

“…As a result, the anchor Lys216 has largely returned to its resting conformation. This prompt restoration of the resting conformation is found important to the pumping mechanism of bR presented in the companion paper (Ren, 2021). Such a lack of substantial change between the ground state and the intermediate I was previously noted by a comparison of the native retinal to a chemically locked C 13 =C 14 double bond (Zhong et al, 1996).…”

Section: Resultsmentioning

confidence: 66%

“…1b inset). The functional implication of these ultrafast SB U-turns is presented in the companion paper (Ren, 2021).…”

Section: Resultsmentioning

confidence: 99%

“…Double bonds are shaded in gray. Intermediates K and L are taken from the companion paper (Ren, 2021). 1) Atomic displacements of each intermediate from the resting state show greater changes in the proximal segment around the SB (top panel).…”

Section: Resultsmentioning

confidence: 99%

“…1c 2 nd panel). This corner and its interaction with helix C is important to the function of this proton pump (Ren, 2021), which is a topic outside the scope of isomerization sampling here. The refined geometry of the retinal in I’ retains a near perfect all- trans configuration, including the Schiff base (SB) double bond C 15 =N ζ , while various single bonds along the polyene chain deviate from the standard anti conformation significantly (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These structures of "pure" photoproducts at atomic resolution reveal widespread conformational changes in all seven helices prior to the all-trans to 13-cis isomerization and after its completion, suggesting that isomerization sampling takes place in bR, where rapid photoisomerizations and single bond rotations are attempted everywhere along the polyene chain of the retinal before the only successful one flips the SB at ~500 fs. The implication of these findings to the proton pumping and directional conductance is presented in a companion paper (Ren, 2021).…”

Section: Significance Statement Introductionmentioning

confidence: 95%

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Photoinduced Isomerization Sampling of Retinal in Bacteriorhodopsin

Ren

¹

2021

Preprint

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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.

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Photoinduced isomerization sampling of retinal in bacteriorhodopsin

Ren

¹

2022

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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 together 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. Significance Statement Photoisomerization of retinal is a critical rearrangement reaction in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. It has been a major challenge to visualize rapid molecular events preceding and following photoisomerization so that many protein functions depending on such reaction remain vaguely understood. Here I report a direct observation of the stereoselectivity of bacteriorhodopsin hence delineate the structural mechanism of isomerization. Upon a light-induced charge separation, the retinal in a flat conformation attempts to perform double bond isomerization and single bond rotation everywhere along its polyene chain before it proceeds to the specific, productive configuration. This observation improves our understanding on how a non-specific attraction force could drive a specific isomerization.

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Spin-Coupled Electron Densities of Iron-Sulfur Cluster Imaged byIn SituSerial Laue Diffraction

Ren

¹

,

Zhang

²

,

Kang

³

et al. 2023

Preprint

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Iron-sulfur clusters are inorganic cofactors found in many proteins involved in fundamental biological processes including DNA processing. The prokaryotic DNA repair enzyme PhrB, a member of the protein family of cryptochromes and photolyases, carries a four-iron-four-sulfur cluster [4Fe4S] in addition to the catalytic cofactor flavin adenine dinucleotide (FAD) and a second pigment 6,7-dimethyl-8-ribityllumazine (DMRL). The light-induced redox reactions of this multi-cofactor protein complex were recently shown as two interdependent photoreductions of FAD and DMRL mediated by the [4Fe4S] cluster functioning as an electron cache to hold a fine balance of electrons. Here, we apply the more traditional temperature-scan cryo-trapping technique in protein crystallography and the newly developed technology of in situ serial Laue diffraction at room temperature. These diffraction methods in dynamic crystallography enable us to capture strong signals of electron density changes in the [4Fe4S] cluster that depict quantized electronic movements. The mixed valence layers of the [4Fe4S] cluster due to spin coupling and their dynamic responses to light illumination are observed directly in our difference maps between its redox states. These direct observations of the quantum effects in a protein bound iron-sulfur cluster have thus opened a window into the mechanistic understanding of metal clusters in biological systems.

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

Cited by 3 publications

References 144 publications

Photoinduced Isomerization Sampling of Retinal in Bacteriorhodopsin

Photoinduced Isomerization Sampling of Retinal in Bacteriorhodopsin

Photoinduced isomerization sampling of retinal in bacteriorhodopsin

Spin-Coupled Electron Densities of Iron-Sulfur Cluster Imaged byIn SituSerial Laue Diffraction

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