Rhodopsins constitute a broad class of retinal-binding photoreceptors. Microbial rhodopsins are canonically activated through an all-trans to 13-cis photoisomerization, whereas animal rhodopsins are mostly activated through an 11-cis to all-trans isomerization. Bestrhodopsins constitute a special microbial rhodopsin subfamily, with bistable rhodopsin domains that can be photoswitched between a far red-absorbing state D661 and a green-absorbing state P540. Its photochemistry involves a peculiar all-trans to 11-cis isomerization for the D661 to P540 photoreaction and vice versa. Here, we present the P. antarctica bestrhodopsin 11-cis to all-trans photoreaction as determined by femtosecond-to-submillisecond transient absorption, femtosecond stimulated Raman and flash-photolysis spectroscopy. The primary photoreaction involves ultrafast isomerizations in 240 fs from the 11-cis reactant to a mixture of highly distorted all-trans and 13-cis photoproducts. The 13-cis fraction then thermally isomerizes to a distorted all-trans RSB in 120 ps. We propose bicycle pedal models for the branched photoisomerizations with corotation of the C11═C12 and C13═C14 double bonds. One reactant fraction undergoes bicycle pedal motion aborted at the C13═C14 double bond, resulting in all-trans retinal. The other fraction undergoes a full bicycle pedal motion of both C11═C12 and C13═C14, resulting in 13-cis retinal. The primary products are trapped high up the ground-state potential energy surface with a low energetic barrier that facilitates thermal isomerization from 13-cis to all-trans retinal in 120 ps. All-trans retinal then structurally and energetically relaxes with subsequent time constants of 0.7 and 62 μs and 4.4 ms, along with counterion protonation, completing the P540 to D661 photoreaction.

• MeSH
• Photochemical Processes MeSH
• Isomerism MeSH
• Retinaldehyde * chemistry   MeSH
• Rhodopsins, Microbial * chemistry   MeSH
• Stereoisomerism MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Retinaldehyde * MeSH
• Rhodopsins, Microbial * MeSH

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

• Keywords
• conical intersection, far-red absorbing rhodopsin, femtosecond stimulated Raman spectroscopy, femtosecond to millisecond spectroscopy, multiple retinal isomerization,
• MeSH
• Diterpenes * MeSH
• Isomerism MeSH
• Protein Conformation MeSH
• Retinaldehyde * chemistry   MeSH
• Rhodopsin * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 13-cis-retinal MeSH Browser
• Diterpenes * MeSH
• Retinaldehyde * MeSH
• Rhodopsin * MeSH

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

• Publication type
• Journal Article MeSH

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

• Publication type
• Journal Article MeSH

Microbial rhodopsins constitute a key protein family in optobiotechnological applications such as optogenetics and voltage imaging. Spectral tuning of rhodopsins into the deep-red and near-infrared spectral regions is of great demand in such applications because more bathochromic light into the near-infrared range penetrates deeper in living tissue. Recently, retinal analogues have been successfully used in ion transporting and fluorescent rhodopsins to achieve red-shifted absorption, activity, and emission properties. Understanding their photochemical mechanism is essential for further design of appropriate retinal analogues but is yet only poorly understood for most retinal analogue pigments. Here, we report the photoreaction dynamics of red-shifted analogue pigments of the proton pump proteorhodopsin (PR) containing A2 (all- trans-3,4-dehydroretinal), MOA2 (all- trans-3-methoxy-3,4-dehydroretinal), or DMAR (all- trans-3-dimethylamino-16-nor-1,2,3,4-didehydroretinal), utilizing femto- to submillisecond transient absorption spectroscopy. We found that the A2 analogue photoisomerizes in 1.4, 3.0, and/or 13 ps upon 510 nm light illumination, which is comparable to the native retinal (A1) in PR. On the other hand, the deprotonation of the A2 pigment Schiff base was observed with a dominant time constant of 67 μs, which is significantly slower than the A1 pigment. In the MOA2 pigment, no isomerization or photoproduct formation was detected upon 520 nm excitation, implying that all the excited molecules returned to the initial ground state in 2.0 and 4.2 ps. The DMAR pigment showed very slow excited state dynamics similar to the previously studied MMAR pigment, but only very little photoproduct was formed. The low efficiency of the photoproduct formation likely is the reason why DMAR analogue pigments of PR showed very weak proton pumping activity.

• MeSH
• Retinaldehyde analogs & derivatives   radiation effects   MeSH
• Rhodopsins, Microbial chemistry   radiation effects   MeSH
• Light MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• proteorhodopsin MeSH Browser
• Retinaldehyde MeSH
• Rhodopsins, Microbial MeSH