scholarly journals Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Keiichi Kojima ◽  
Susumu Yoshizawa ◽  
Masumi Hasegawa ◽  
Masaki Nakama ◽  
Marie Kurihara ◽  
...  
Keyword(s):  
Schiff Base ◽  
Proton Pump ◽  
Flash Photolysis ◽  
Blue Shift ◽  
Spectroscopic Studies ◽  
Spectral Shift ◽  
Bulk Solution ◽  
Proton Pumps ◽  
Ph Titration ◽  
Retinal Chromophore

AbstractThe photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named ‘schizorhodopsin (SzR)’ has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pKa = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pKa = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral ‘blue-shift’ due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

Structural Factors Determining the Absorption Spectrum of Channelrhodopsins: A Case Study of the Chimera C1C2

2020 ◽  
Author(s):  
Suliman Adam ◽  
Christian Wiebeler ◽  
Igor Schapiro
Keyword(s):  
Hydrogen Bonding ◽  
Absorption Spectrum ◽  
Schiff Base ◽  
Absorption Maximum ◽  
Rational Design ◽  
Blue Shift ◽  
Spectral Shift ◽  
Structural Basis ◽  
Neural Cells ◽  
Structural Factors

Channelrhodopsins are photosensitive proteins that trigger flagella motion in single cell algae and have been successfully utilized in optogenetic applications. In optogenetics light is used to activate neural cells in living organisms, which can be achieved by exploiting the ion channel signaling of channelrhodopsins. Tailoring channelrhodopsins for such applications includes the tuning of the absorption maximum. In order to establish rational design and to obtain a desired spectral shift, a basic understanding of the absorption spectrum is required. We have studied the chimera C1C2 as a representative of this protein family and the first member with an available crystal structure. For this purpose, we sampled the conformations of C1C2 using QM/MM molecular dynamics, and subjected the resulting snapshots of the trajectory to excitation energy calculations using ADC(2) and simplified TD-DFT. In contrast to previous reports, we found that different hydrogen-bonding networks—involving the retinal protonated Schiff base, the putative counterions E162 and D292 as well as water molecules—had only a small impact on the absorption spectrum. However, in case of deprotonated E162 increasing the distance to the Schiff base hydrogen-bonding partner led to a systematic blue shift. The β-ionone ring rotation was identified as another important contributor. Yet the most important factor was found to be the bond length alternation and bond order alternation that were linearly correlated to the absorption maximum by up to 62 % and 82 %, respectively. We ascribe this novel insight into the structural basis of the absorption spectrum to our enhanced protein setup that includes membrane embedding as well as long and extensive sampling.

scholarly journals Structural Factors Determining the Absorption Spectrum of Channelrhodopsins: A Case Study of the Chimera C1C2

2020 ◽  
Author(s):  
Suliman Adam ◽  
Christian Wiebeler ◽  
Igor Schapiro
Keyword(s):  
Hydrogen Bonding ◽  
Absorption Spectrum ◽  
Schiff Base ◽  
Absorption Maximum ◽  
Rational Design ◽  
Blue Shift ◽  
Spectral Shift ◽  
Structural Basis ◽  
Neural Cells ◽  
Structural Factors

Channelrhodopsins are photosensitive proteins that trigger flagella motion in single cell algae and have been successfully utilized in optogenetic applications. In optogenetics light is used to activate neural cells in living organisms, which can be achieved by exploiting the ion channel signaling of channelrhodopsins. Tailoring channelrhodopsins for such applications includes the tuning of the absorption maximum. In order to establish rational design and to obtain a desired spectral shift, a basic understanding of the absorption spectrum is required. We have studied the chimera C1C2 as a representative of this protein family and the first member with an available crystal structure. For this purpose, we sampled the conformations of C1C2 using QM/MM molecular dynamics, and subjected the resulting snapshots of the trajectory to excitation energy calculations using ADC(2) and simplified TD-DFT. In contrast to previous reports, we found that different hydrogen-bonding networks—involving the retinal protonated Schiff base, the putative counterions E162 and D292 as well as water molecules—had only a small impact on the absorption spectrum. However, in case of deprotonated E162 increasing the distance to the Schiff base hydrogen-bonding partner led to a systematic blue shift. The β-ionone ring rotation was identified as another important contributor. Yet the most important factor was found to be the bond length alternation and bond order alternation that were linearly correlated to the absorption maximum by up to 62 % and 82 %, respectively. We ascribe this novel insight into the structural basis of the absorption spectrum to our enhanced protein setup that includes membrane embedding as well as long and extensive sampling.

scholarly journals Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates

2021 ◽  
Vol 8 ◽  
Author(s):  
Sabine Panzer ◽  
Chong Zhang ◽  
Tilen Konte ◽  
Celine Bräuer ◽  
Anne Diemar ◽  
...  
Keyword(s):  
Proton Pump ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Aureobasidium Pullulans ◽  
Green Light ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Proton Pumps ◽  
C Terminus ◽  
Pump Activity

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

scholarly journals Breaking the Carboxyl Rule

2013 ◽  
Vol 288 (29) ◽  
pp. 21254-21265 ◽  
Author(s):  
Sergei P. Balashov ◽  
Lada E. Petrovskaya ◽  
Eleonora S. Imasheva ◽  
Evgeniy P. Lukashev ◽  
Andrei K. Dioumaev ◽  
...  
Keyword(s):  
Schiff Base ◽  
Water Molecule ◽  
Proton Pump ◽  
Rate Constants ◽  
Proton Donor ◽  
Carboxyl Group ◽  
Amino Group ◽  
Wild Type ◽  
Fast Phase ◽  
Proton Uptake

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

The vacuolar proton pump of Dictyostelium discoideum: molecular cloning and analysis of the 100 kDa subunit

1996 ◽  
Vol 109 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
T. Liu ◽  
M. Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Proton Pump ◽  
Single Gene ◽  
Consensus Sequence ◽  
Amino Acid Sequences ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Variable Regions ◽  
Endosomal Membranes ◽  
Vacuolar Proton Pump

The vacuolar proton pump is a highly-conserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryotic cells. Its largest subunit (95-116 kDa) occurs in tissue and organelle-specific isoforms and thus may be involved in targeting the enzyme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100 kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessu et al. (1995) J. Cell Sci. 108, 3331–3337) match the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells.

Altered expression of the 100 kDa subunit of the Dictyosteliumvacuolar proton pump impairs enzyme assembly, endocytic function and cytosolic pH regulation

2002 ◽  
Vol 115 (9) ◽  
pp. 1907-1918 ◽  
Author(s):  
Tongyao Liu ◽  
Christian Mirschberger ◽  
Lilian Chooback ◽  
Quyen Arana ◽  
Zeno Dal Sacco ◽  
...  
Keyword(s):  
Proton Pump ◽  
Catalytic Domain ◽  
Ph Regulation ◽  
Proton Pumps ◽  
Cytosolic Ph ◽  
Acid Media ◽  
Dynamic Regulation ◽  
Atpase Subunit ◽  
Altered Expression ◽  
Mutant Cells

The vacuolar proton pump (V-ATPase) appears to be essential for viability of Dictyostelium cells. To investigate the function of VatM, the 100 kDa transmembrane V-ATPase subunit, we altered its level. By means of homologous recombination, the promoter for the chromosomal vatM gene was replaced with the promoter for the act6 gene, yielding the mutant strain VatMpr. The act6 promoter is much more active in cells growing axenically than on bacteria. Thus, transformants were selected under axenic growth conditions, then shifted to bacteria to determine the consequences of reduced vatM expression. When VatMpr cells were grown on bacteria,the level of the 100 kDa V-ATPase subunit dropped, cell growth slowed, and the A subunit, a component of the peripheral catalytic domain of the V-ATPase,became mislocalized. These defects were complemented by transformation of the mutant cells with a plasmid expressing vatM under the control of its own promoter. Although the principal locus of vacuolar proton pumps in Dictyostelium is membranes of the contractile vacuole system, mutant cells did not manifest osmoregulatory defects. However, bacterially grown VatMpr cells did exhibit substantially reduced rates of phagocytosis and a prolonged endosomal transit time. In addition, mutant cells manifested alterations in the dynamic regulation of cytosolic pH that are characteristic of normal cells grown in acid media, which suggested that the V-ATPase also plays a role in cytosolic pH regulation.

Interaction of SO2 with unsaturated Schiff-base complexes; thermal, magnetic, and spectroscopic studies

2009 ◽  
Vol 62 (20) ◽  
pp. 3377-3383 ◽  
Author(s):  
Akila A. Saleh ◽  
Abdel Razak M. Tawfik ◽  
Mosad A. El Ghamry ◽  
Samy M. Abu-el-wafa
Keyword(s):  
Schiff Base ◽  
Spectroscopic Studies ◽  
Schiff Base Complexes

Components and mechanism of action of ATP-driven proton pomps

1979 ◽  
Vol 57 (12) ◽  
pp. 1351-1358 ◽  
Author(s):  
Marcelo Alfonzo ◽  
Efraim Racker
Keyword(s):  
Electron Microscopy ◽  
Electron Micrographs ◽  
Proton Pump ◽  
Mechanism Of Action ◽  
Proton Channel ◽  
Proton Pumps ◽  
Positive Staining ◽  
Freeze Etching ◽  
Opening And Closing ◽  
Exchange Activity

We have studied the composition of ATP-driven proton pumps from bovine heart mitochondria and have reconstituted the oligomycin-sensitive ATPase complex from its individual components. The complex contains 9 to 10 subunits of which 5 are assembled in the soluble F1 protein, 2 are required for the attachment of F1 to the membrane and 2 form the proton channel within the membrane. With the help of information obtained from studies of the chloroplast and the bacterial proton pumps, we can tentatively assign a function to each of the subunits of the pump. The position of F1 outside of the membrane seen in electron micrographs of negatively stained preparations, does not appear to be an artifact. Evidence from immunological studies, chemical derivatizations as well as further electron microscopy (positive staining and freeze–etching), support this statement. We describe in this paper a 28 000-dalton polypeptide which has been isolated from the mitochondria membrane and is required for the reconstitution of oligomycin-sensitive ATPase and 32Pi–ATP exchange activity. We propose a mechanism of action of the proton pump in which the key energy-yielding reaction is the binding of Mg2+ to the protein. The function of the proton gradient is to displace Mg2+ from this site to permit cyclic repetition of the binding process. Essential for this scheme is the cyclic opening and closing of the proton channel. We have outlined our present approaches to test this hypothesis.

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