scholarly journals Proton transfer pathway in anion channelrhodopsin-1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Masaki Tsujimura ◽  
Keiichi Kojima ◽  
Shiho Kawanishi ◽  
Yuki Sudo ◽  
Hiroshi Ishikita
Keyword(s):  
Schiff Base ◽  
Proton Transfer ◽  
Hek293 Cells ◽  
Wild Type ◽  
Gating Mechanism ◽  
Guillardia Theta ◽  
In The Wild ◽  
Mechanical Approach ◽  
Dynamics Simulations ◽  
Original Crystal

Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

scholarly journals Proton-mediated gating mechanism of anion channelrhodopsin-1

2021 ◽  
Author(s):  
Masaki Tsujimura ◽  
Keiichi Kojima ◽  
Shiho Kawanishi ◽  
Yuki Sudo ◽  
Hiroshi Ishikita
Keyword(s):  
Schiff Base ◽  
Hek293 Cells ◽  
Wild Type ◽  
Conducting Channel ◽  
Gating Mechanism ◽  
Guillardia Theta ◽  
In The Wild ◽  
Mechanical Approach ◽  
Dynamics Simulations ◽  
Original Crystal

Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths were also analyzed, using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shortens and lengthens the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients towards the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

scholarly journals Photochemical reaction cycle transitions during anion channelrhodopsin gating

2016 ◽  
Vol 113 (14) ◽  
pp. E1993-E2000 ◽  
Author(s):  
Oleg A. Sineshchekov ◽  
Hai Li ◽  
Elena G. Govorunova ◽  
John L. Spudich
Keyword(s):  
Schiff Base ◽  
Hek293 Cells ◽  
Proton Acceptor ◽  
Channel Activity ◽  
Wild Type ◽  
Anion Selectivity ◽  
Slow Channel ◽  
Guillardia Theta ◽  
Report Analysis ◽  
Ion Conducting

A recently discovered family of natural anion channelrhodopsins (ACRs) have the highest conductance among channelrhodopsins and exhibit exclusive anion selectivity, which make them efficient inhibitory tools for optogenetics. We report analysis of flash-induced absorption changes in purified wild-type and mutant ACRs, and of photocurrents they generate in HEK293 cells. Contrary to cation channelrhodopsins (CCRs), the ion conducting state of ACRs develops in an L-like intermediate that precedes the deprotonation of the retinylidene Schiff base (i.e., formation of an M intermediate). Channel closing involves two mechanisms leading to depletion of the conducting L-like state: (i) Fast closing is caused by a reversible L⇔M conversion. Glu-68 in Guillardia theta ACR1 plays an important role in this transition, likely serving as a counterion and proton acceptor at least at high and neutral pH. Incomplete suppression of M formation in the GtACR1_E68Q mutant indicates the existence of an alternative proton acceptor. (ii) Slow closing of the channel parallels irreversible depletion of the M-like and, hence, L-like state. Mutation of Cys-102 that strongly affected slow channel closing slowed the photocycle to the same extent. The L and M intermediates were in equilibrium in C102A as in the WT. In the position of Glu-123 in channelrhodopsin-2, ACRs contain a noncarboxylate residue, the mutation of which to Glu produced early Schiff base proton transfer and strongly inhibited channel activity. The data reveal fundamental differences between natural ACR and CCR conductance mechanisms and their underlying photochemistry, further confirming that these proteins form distinct families of rhodopsin channels.

Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

2001 ◽  
Vol 359 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Valeria MENCHISE ◽  
Catherine CORBIER ◽  
Claude DIDIERJEAN ◽  
Michele SAVIANO ◽  
Ettore BENEDETTI ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Chlamydomonas Reinhardtii ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Careful Analysis ◽  
Wild Type ◽  
Thioredoxin H ◽  
Dynamics Simulations

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

The effect of pressure on the excited-state proton transfer in the wild-type green fluorescent protein

2008 ◽  
Vol 455 (4-6) ◽  
pp. 303-306 ◽  
Author(s):  
Pavel Leiderman ◽  
Dan Huppert ◽  
S. James Remington ◽  
Laren M. Tolbert ◽  
Kyril M. Solntsev
Keyword(s):  
Excited State ◽  
Green Fluorescent Protein ◽  
Proton Transfer ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Effect Of Pressure ◽  
Excited State Proton Transfer ◽  
In The Wild ◽  
Green Fluorescent

scholarly journals Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form

2015 ◽  
Vol 9 ◽  
pp. BBI.S25626 ◽  
Author(s):  
Khadija Amine ◽  
Lamia Miri ◽  
Adil Naimi ◽  
Rachid Saile ◽  
Abderrahmane El Kharrim ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Active Site ◽  
Paraoxonase 1 ◽  
Wild Type ◽  
Repetitive Movement ◽  
Catalytic Core ◽  
In The Wild ◽  
Dynamics Simulations ◽  
L1 And L2 ◽  
The Impact

There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme.

scholarly journals Origin of Conformational Dynamics in a Globular Protein

2019 ◽  
Author(s):  
Adam M. Damry ◽  
Marc M. Mayer ◽  
Aron Broom ◽  
Natalie K. Goto ◽  
Roberto A. Chica
Keyword(s):  
Conformational Dynamics ◽  
Protein Structures ◽  
Vital Role ◽  
Globular Protein ◽  
Conformational Exchange ◽  
Solution Nmr ◽  
Wild Type ◽  
In The Wild ◽  
Dynamics Simulations ◽  
The Impact

AbstractProtein structures are dynamic, undergoing specific motions that can play a vital role in function. However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual substitutions of core residues in DANCER-3, a streptococcal protein G domain β1 (Gβ1) variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native Gβ1 structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how conformational dynamics can appear in a stable globular fold, a critical step in the molecular evolution of new dynamics-linked functions.

KCNQ2-Associated Neonatal Epilepsy: Phenotype Might Correlate With Genotype

2017 ◽  
Vol 32 (8) ◽  
pp. 704-711 ◽  
Author(s):  
Inn-Chi Lee ◽  
Jiann-Jou Yang ◽  
Jao-Shwann Liang ◽  
Tung-Ming Chang ◽  
Shuan-Yow Li
Keyword(s):  
Patch Clamp ◽  
Seizure Frequency ◽  
Clinical Phenotype ◽  
Hek293 Cells ◽  
The Novel ◽  
Wild Type ◽  
Neurodevelopmental Outcomes ◽  
Whole Cell Patch Clamp ◽  
In The Wild ◽  
Type Gene

We analyzed the KCNQ2 wild-type gene and 3 mutations to highlight the important association between the KCNQ2 phenotype and genotype. The clinical phenotypes of 3 mutations (p.E515D, p.V543 M, and p.R213Q) were compared. KCNQ2, wild-type, and mutant KCNQ2 alleles were transfected into HEK293 cells before whole-cell patch-clamp analysis. Neurodevelopmental outcomes were worst in patients with the p.R213Q mutation, better in patients with the p.E515D mutation, and best in patients with the novel p.V543 M mutation. The currents in p.E515D and in p.V543 M were significantly lower than in the wild type in homomeric and heteromeric transfected HEK293 cells ( P < .05). The opening threshold shifted to values that were more positive, and the maximal current induced by strong depolarization was higher in cells with the p.E515D and p.R213Q mutations. We provide evidence that genotype is involved in determining clinical phenotype, including the seizure frequency and outcome.

scholarly journals The Impact of Mutation L138F/L210F on the Orai Channel: A Molecular Dynamics Simulation Study

2021 ◽  
Vol 8 ◽  
Author(s):  
Xiaoqian Zhang ◽  
Hua Yu ◽  
Xiangdong Liu ◽  
Chen Song
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Calcium Release ◽  
Hydrophobic Interactions ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Hydrophobic Region ◽  
In The Wild ◽  
Dynamics Simulations ◽  
The Impact

The calcium release-activated calcium channel, composed of the Orai channel and the STIM protein, plays a crucial role in maintaining the Ca2+ concentration in cells. Previous studies showed that the L138F mutation in the human Orai1 creates a constitutively open channel independent of STIM, causing severe myopathy, but how the L138F mutation activates Orai1 is still unclear. Here, based on the crystal structure of Drosophila melanogaster Orai (dOrai), molecular dynamics simulations for the wild-type (WT) and the L210F (corresponding to L138F in the human Orai1) mutant were conducted to investigate their structural and dynamical properties. The results showed that the L210F dOrai mutant tends to have a more hydrated hydrophobic region (V174 to F171), as well as more dilated basic region (K163 to R155) and selectivity filter (E178). Sodium ions were located deeper in the mutant than in the wild-type. Further analysis revealed two local but essential conformational changes that may be the key to the activation. A rotation of F210, a previously unobserved feature, was found to result in the opening of the K163 gate through hydrophobic interactions. At the same time, a counter-clockwise rotation of F171 occurred more frequently in the mutant, resulting in a wider hydrophobic gate with more hydration. Ultimately, the opening of the two gates may facilitate the opening of the Orai channel independent of STIM.

scholarly journals Neonates with the KCNQ2 Y755C Variants: Not Associated with Neonatal Epileptic Encephalopathy

2021 ◽  
Vol 4 (3) ◽  
Keyword(s):  
Electrical Current ◽  
Epileptic Encephalopathy ◽  
Hek293 Cells ◽  
Functional Study ◽  
Neonatal Seizure ◽  
Pediatric Epilepsy ◽  
Wild Type ◽  
Functional Changes ◽  
Investigation Methods ◽  
In The Wild

Background: Pediatric epilepsy caused by a KCNQ2 gene mutation usually manifests the phenotype of a neonatal seizure. KCNQ2 encephalopathy in newborns continues to be reported on. Objectives: The exact mechanism and phenotype of the KCNQ2 mutation still require investigation. Methods: One hundred twenty-one patients with childhood epilepsy without an identified cause underwent KCNQ2 sequencing. KCNQ2 mutation variants were transfected into human embryonic kidney 293 (HEK293) cells to investigate functional changes. Results: Two patients with the c.2264G>G/A (p.Y755C) variant had neonatal epileptic encephalopathy: one had electroencephalography (EEG) burst suppression and the other had multiple focal spikes. However, the mutation was not found in the 80 healthy adult claiming without ever seizures before. A functional study showed that p.Y755C currents were not different from those in the wild-type and from those in the benign (p.N780T) polymorphism in homomeric and heteromeric (wild-type KCNQ2: mutant = 1:1) transfected HEK293 cells. Electrical current differences between HEK293 cells with wildtype mutations and cells transfected with the wild-type KCNQ2, KCNQ3, and p.Y755C mutations in a 1:2:1 ratio were not significant. Their seizures remitted after they turned 1 year old. Conclusion: We suggest that patients with the KCNQ2 p.Y755C mutations are not associated with neonatal epileptic encephalopathy

scholarly journals Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
George Khelashvili ◽  
Anoop Narayana Pillai ◽  
Joon Lee ◽  
Kalpana Pandey ◽  
Alexander M. Payne ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Retinitis Pigmentosa ◽  
Molecular Dynamics Simulations ◽  
Autosomal Dominant ◽  
Hek293 Cells ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Dynamics Simulations ◽  
G Protein Coupled

AbstractMutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

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