The Early Steps in the Photocycle of a Photosensor Protein Sensory Rhodopsin I from Salinibacter ruber

Abstract

Light absorption by the photoreceptor microbial rhodopsin triggers trans-cis isomerization of the retinal chromophore surrounded by seven transmembrane α-helices. Sensory rhodopsin I (SRI) is a dual functional photosensory rhodopsin both for positive and negative phototaxis in microbes. By making use of the highly stable SRI protein from Salinibacter ruber (SrSRI), the early steps in the photocycle were studied by time-resolved spectroscopic techniques. All of the temporal behaviors of the Sn←S1 absorption, ground-state bleaching, K intermediate absorption, and stimulated emission were observed in the femto- to picosecond time region by absorption spectroscopy. The primary process exhibited four dynamics similar to other microbial rhodopsins. The first dynamics (τ1 ∼ 54 fs) corresponds to the population branching process from the Franck-Condon region to the reactive (S1(r)) and nonreactive (S1(nr)) S1 states. The second dynamics (τ2 = 0.64 ps) is the isomerization process of the S1(r) state to generate the ground-state 13-cis form, and the third dynamics (τ3 = 1.8 ps) corresponds to the internal conversion of the S1(nr) state. The fourth component (τ3' = 2.5 ps) is assignable to the J-decay (K-formation). This reaction scheme was further supported by the results of fluorescence spectroscopy. To investigate the protein response(s), the spectral changes of the tryptophan bands were monitored by ultraviolet resonance Raman spectroscopy. The intensity change following the K formation in the chromophore structure (τ ∼ 17 ps) was significantly small in SrSRI as compared with other microbial rhodopsins. We also analyzed the effect(s) of Cl(-) binding on the ultrafast dynamics of SrSRI. Compared with a chloride pump Halorhodopsin, Cl(-) binding to SrSRI was less effective for the excited-state dynamics, whereas the binding altered the structural changes of tryptophan following the K-formation, which was the characteristic feature for SrSRI. On the basis of these results, a primary photoreaction scheme of SrSRI together with the role of chloride binding is proposed.

Demonstration of a Light-Driven SO42– Transporter and Its Spectroscopic Characteristics

“…time region (~10 ps) only in the presence of NaCl. This spectral change is attributable to the conversion from the J-intermediate to the K-intermediate, which has been well characterized for other rhodopsins 54,57,[60][61][62]. The corresponding blue-shift was not recognized under the other salt conditions.…”

“…The shortest lifetime component (τ 0, ~50 fs) was attributable to the blue-shift of the ESA band and the red-shift of the SE band, as previously reported for other rhodopsins. 52,53,54,55 According to these previous studies, we assigned the τ 0 component to the initial relaxation from the Franck-Condon state to the relaxed S 1 state. After these spectral shifts, the ESA and SE bands concomitantly decay with the time constants of τ 1 , τ 2 and τ 3 .…”

“…Such multi-exponential behavior of the S 1 decay has been observed for other rhodopsins as well. 53,54,56,57 Although its origin is still under debate, 58 it has been proposed that some rhodopsins have structural heterogeneity in the ground state; multiple species that have slightly different environments around the chromophore are present in the sample. 59 These multiple species can be excited in parallel, and the generated S 1 states individually decay with different lifetimes reflecting different environments around the chromophore.…”

An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli

“…A member of the 7 transmembrane (TM) domain family of proteins, rhodopsins have vitamin-A aldehyde retinal as their chromophore, in which a specific lysine residue located in the middle of the TM region binds to a retinal molecule resulting in formation of the protonated Schiff base linkage 1 2 3 . The photoabsorption of the retinal in rhodopsins triggers the stepwise reactions that accompany the trans-cis photoisomerization upon formation of the early photointermediate 3 4 . Rhodopsin molecules are roughly classified into two groups, microbial type-1 rhodopsins and animal type-2 rhodopsins 1 5 .…”

Time-Resolved Resonance Raman Spectroscopy and Application to Studies on Ultrafast Protein Dynamics

“…The photoisomerization of the chromophore takes place in subpicoseconds, 9497 which induces sequential changes in the protein structure that are important for protein functions. To examine protein responses to the chromophore isomerization, time-resolved UVRR spectra of five types of microbial rhodopsins, BR, 98 halorhodopsin (HR), 99 sensory rhodopsin I (SRI), 100 sensory rhodopsin II (SRII), 101 and Anabaena sensory rhodopsin (ASR), 102 were obtained. In this subsection, I summarize our recent studies on the primary protein response of the microbial rhodopsins associated with photoreactions of the chromophore.…”