Fluorescence spectra of bacteriorhodopsin and the intermediates O and Q at room temperature

Ohtani, Hiroyuki; Tsukamoto, Yasuhisa; Sakoda, Yuusaku; Hamaguchi, Hiro‐o

doi:10.1016/0014-5793(94)01440-c

Cited by 18 publications

(22 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fluorescence of Arch was initially believed to result from the ground state, but instead was found to be the result of a state formed from the N state by exposure to yellow light, called the Q state. A similar Q state has also been observed in bR due to the sequential absorption of 3 photons (Ohtani et al, 1992 , 1995 ). While green light is sufficient to create Q from N, orange light is required for excitation of Q.…”

Section: Discussionsupporting

confidence: 67%

Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS)

Nadeau

2015

Front. Neurosci.

View full text Add to dashboard Cite

Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS), which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanosecond time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested.

show abstract

Section: Discussionsupporting

confidence: 67%

Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS)

Nadeau

2015

Front. Neurosci.

View full text Add to dashboard Cite

show abstract

“…It has been reported that absorption of a photon by the N-intermediate of the native BR photocycle leads to formation of a “Q” intermediate which exhibits fluorescence emission above >700 nm 67 . Hence, the fluorescence observed for D95N is expected to be a highly non-linear 3-photon process involving a first photon absorbed by O all trans , a second photon absorbed by N 13- cis and third photon which excites the Q intermediate to emit a fluorescent photon (this is consistent with recent studies (A. Cohen and J. Kralj, private communication)).…”

Section: Discussionmentioning

confidence: 99%

Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

et al. 2012

View full text Add to dashboard Cite

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3–11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (Oall-trans) is found. At higher pH a second 13-cis retinal N-like species (N13-cis) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N13-cis detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N13-cis to M13-cis levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.

show abstract

“…In this regard, an earlier study of native AR3 based on measuring photogenerated intermediates in E. coli and HEK293 single cells ( 84 ) concluded that in this case the fluorescence detected in the NIR involves a 3‐photon absorption process. The first photon absorbed by the ground state of AR3 which has a maximum absorbance wavelength near 570 nm generates the transient N photocycle intermediate which then upon photon absorption forms a previously identified NIR fluorescent Q species (note this is different from Q 380 state discussed previously in conjunction with pink membrane) ( 85 ). However, it is likely that the O‐like ground states of QuasAr2 and NovArch are distinct from the ground state of native AR3 which has a negatively charged SB counterion.…”

Section: Discussionmentioning

confidence: 95%

Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors

Mei

Cavini

Mamaeva

et al. 2021

Photochem & Photobiology

View full text Add to dashboard Cite

Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better understand the photophysical properties of OTVSs, we used a combination of UV-Vis absorption, fluorescence and FT-Raman spectroscopy to characterize Qua-sAr2 and NovArch, two closely related mutants derived from the proton pump archaerhodopsin-3 (AR3). We find both QuasAr2 and NovArch can be optically cycled repeatedly between O-like and M-like states using 5-min exposure to red (660 nm) and near-UV (405 nm) light. Longer red-light exposure resulted in the formation of a long-lived photoproduct similar to pink membrane, previously found to be a photoproduct of the BR O intermediate with a 9-cis retinylidene chromophore configuration. However, unlike QuasAr2 whose O-like state is stable in the dark, NovArch exhibits an O-like state which slowly partially decays in the dark to a stable Mlike form with a deprotonated Schiff base and a 13-cis,15-anti retinylidene chromophore configuration. These results reveal a previously unknown complexity in the photochemistry of OTVSs including the ability to optically switch between different long-lived states. The possible molecular basis of these newly discovered properties along with potential optogenetic and biotechnological applications are discussed.

show abstract

Fluorescence spectra of bacteriorhodopsin and the intermediates O and Q at room temperature

Cited by 18 publications

References 20 publications

Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS)

Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS)

Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors

Contact Info

Product

Resources

About