Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Lörinczi, Éva; Verhoefen, Mirka-Kristin; Wachtveitl, Josef; Woerner, Andreas C.; Glaubitz, Clemens; Engelhard, Martin; Bamberg, Ernst; Friedrich, Thomas

doi:10.1016/j.jmb.2009.07.055

Cited by 51 publications

(73 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It was shown that the pair Asp-85/His-57 allows the protein to stabilize Asp85 in the unprotonated state in a wide range of pH, which is necessary to keep the proton pump functional (11). However, in proteorhodopsin, where the proton acceptor pair is the same, direction of the proton flow may be reversed at acidic pH (29). Therefore, a priori, Asp-85/His-57 cannot be considered as a sufficient and universal stabilizer of proton pumping in a wide range of pH.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

Gushchin

Chervakov

Kuzmichev

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

View full text Add to dashboard Cite

Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESR's proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 3 10 - and π-helix–like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.

show abstract

Section: Resultsmentioning

confidence: 99%

Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

Gushchin

Chervakov

Kuzmichev

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

View full text Add to dashboard Cite

show abstract

“…Therefore, it is conceivable that the negative transient current is due to a fast and transient proton release towards the “wrong” CP-oriented side, as a consequence of the increased distance to Asp-85 and the already opened CP channel. Similar negative transient current components upon blue laser flash illumination have also been reported for the BR homolog proteorhodopsin (PR) from γ-proteobacteria, although in the latter case, this is not due to an increased distance between the retinal SB and the primary proton acceptor group (Asp-97 in PR), but due to the fact that the pK a of Asp-97 (PR) is around 7.2, so that about 50% of the prooton acceptor groups are protonated at neutral pH [36]. This futile side reaction may contribute to the low proton pumping efficiency, as does the substantially slowed SB reprotonation through the (already open) CP pathway, as judged from the time constants of the photocurrent’s off-response (indicating slowed M decay).…”

Section: Discussionmentioning

confidence: 97%

Voltage Dependence of Proton Pumping by Bacteriorhodopsin Mutants with Altered Lifetime of the M Intermediate

et al. 2013

Self Cite

View full text Add to dashboard Cite

The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum is tightly regulated by the [H+] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L) or stabilized (D96N, D96G) M intermediate in response to green light (to probe H+ pumping) and blue laser flashes (to probe accumulation/decay of M). These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H+ pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H+ pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri) shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations. Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H+ gradient generation.

show abstract

“…Thus reductions in membrane potential slow down the pump, and more light is needed to achieve the same amount of hyperpolarization at more negative membrane potentials. In general, pump direction remains unchanged under all physiologically relevant conditions; the reported “pump inversion” phenomenon observed in PR is due to a leakiness of the pump under extremely negative voltages plus strong pH gradients (Lörinczi et al, 2009). …”

Section: Mechanistic Considerationsmentioning

confidence: 99%

The Microbial Opsin Family of Optogenetic Tools

Zhang

Vierock

Yizhar

et al. 2011

Cell

478

467

View full text Add to dashboard Cite

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

show abstract

Voltage- and pH-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-type and Mutant Proteorhodopsins upon Expression in Xenopus Oocytes

Cited by 51 publications

References 50 publications

Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

Voltage Dependence of Proton Pumping by Bacteriorhodopsin Mutants with Altered Lifetime of the M Intermediate

The Microbial Opsin Family of Optogenetic Tools

Contact Info

Product

Resources

About