Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate

ABSTRACTChannelrhodopsins (ChR) are cation channels that can be expressed heterologously in various living tissues, including cardiac and neuronal cells. To tune spatial and temporal control of potentials across ChR-enriched cell membranes, it is essential to understand how pore hydration impacts the ChR photocycle kinetics. Here, we measure channel opening and closing rates of channelrhodopsin chimera and selected variants (C1C2 wild type, C1C2-N297D, C1C2-N297V, and C1C2-V125L) and correlate them with changes in chemical interactions among functionally important residues in both closed and open states. Kinetic results substantiate that replacement of helices I and II in ChR2 with corresponding residues from ChR1, to make the chimera C1C2, affects the kinetics of channelrhodopsin pore gating significantly, making C1C2 a unique channel. As a prerequisite for studies of ion transport, detailed understanding of the water pathway within a ChR channel is important. Our atomistic simulations confirm that opening of the channel and initial hydration of the previously dry gating regions between helices I, II, III, and VII of the channel occurs with 1) the presence of 13-cis retinal; 2) deprotonation of a glutamic acid gating residue, E129; and 3) subsequent weakening of the central gate hydrogen bond between the same glutamic acid E129 and asparagine N282 in the central region of the pore. Also, an aspartate (D292) is the unambiguous primary proton acceptor for the retinal Schiff base in the hydrated channel.SIGNIFICANCEChannelrhodopsins (ChR) are light-sensitive ion channels used in optogenetics, a technique that applies light to selectively and non-invasively control cells (e.g., neurons) that have been modified genetically to express those channels. Using electrophysiology, we measured the opening and closing rates of a ChR chimera, and several variants, and correlated those rates with changes in chemical interactions determined from atomistic simulations. Significant new insights include correlation of single-point-mutations with four factors associated with pore hydration and cation conductance. Additionally, our work unambiguously identifies the primary proton acceptor for the retinal chromophore in the channel open state. These new insights add to mechanistic understanding of light-gated membrane transport and should facilitate future efforts to control membrane potentials spatially and temporally in optogenetics.

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Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

10.26434/chemrxiv.11481834.v1 ◽

2019 ◽

Author(s):

Nobutaka Fujieda ◽

Miho Yuasa ◽

Yosuke Nishikawa ◽

Genji Kurisu ◽

Shinobu Itoh ◽

...

Keyword(s):

Michael Addition ◽

In Silico ◽

Addition Reaction ◽

Single Point ◽

Point Mutations ◽

Unsaturated Ketone ◽

Michael Addition Reaction ◽

Beta Barrel ◽

Cupin Superfamily ◽

Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

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Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

Biomacromolecules ◽

10.1021/acs.biomac.1c00443 ◽

2021 ◽

Author(s):

Marisa L. Martino ◽

Stephen N. Crooke ◽

Marianne Manchester ◽

M.G. Finn

Keyword(s):

Single Point ◽

Point Mutations ◽

Virus Like Particles ◽

Single Point Mutations

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mCSM-membrane: predicting the effects of mutations on transmembrane proteins

Nucleic Acids Research ◽

10.1093/nar/gkaa416 ◽

2020 ◽

Vol 48 (W1) ◽

pp. W147-W153 ◽

Cited By ~ 6

Author(s):

Douglas E V Pires ◽

Carlos H M Rodrigues ◽

David B Ascher

Keyword(s):

Membrane Proteins ◽

Single Point ◽

Point Mutations ◽

Web Server ◽

Protein Coding ◽

Pathogenic Variants ◽

Model Protein ◽

Matthew’S Correlation Coefficient ◽

Membrane Protein Stability ◽

User Friendly

Abstract Significant efforts have been invested into understanding and predicting the molecular consequences of mutations in protein coding regions, however nearly all approaches have been developed using globular, soluble proteins. These methods have been shown to poorly translate to studying the effects of mutations in membrane proteins. To fill this gap, here we report, mCSM-membrane, a user-friendly web server that can be used to analyse the impacts of mutations on membrane protein stability and the likelihood of them being disease associated. mCSM-membrane derives from our well-established mutation modelling approach that uses graph-based signatures to model protein geometry and physicochemical properties for supervised learning. Our stability predictor achieved correlations of up to 0.72 and 0.67 (on cross validation and blind tests, respectively), while our pathogenicity predictor achieved a Matthew's Correlation Coefficient (MCC) of up to 0.77 and 0.73, outperforming previously described methods in both predicting changes in stability and in identifying pathogenic variants. mCSM-membrane will be an invaluable and dedicated resource for investigating the effects of single-point mutations on membrane proteins through a freely available, user friendly web server at http://biosig.unimelb.edu.au/mcsm_membrane.

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In Vivo Evidence for TonB Dimerization

Journal of Bacteriology ◽

10.1128/jb.185.19.5747-5754.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5747-5754 ◽

Cited By ~ 50

Author(s):

Annette Sauter ◽

S. Peter Howard ◽

Volkmar Braun

Keyword(s):

Single Point ◽

Point Mutations ◽

Ferric Citrate ◽

Site Directed Mutagenesis ◽

Toxin Gene ◽

Transmembrane Region ◽

Dimer Formation ◽

Citrate Transport ◽

Hybrid Proteins

ABSTRACT TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and φ80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

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MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

Biochemical Journal ◽

10.1042/bcj20170357 ◽

2017 ◽

Vol 474 (18) ◽

pp. 3189-3205 ◽

Cited By ~ 6

Author(s):

Ashoka Chary Taviti ◽

Tushar Kant Beuria

Keyword(s):

Cell Division ◽

Single Point ◽

Point Mutations ◽

Direct Interaction ◽

Ring Formation ◽

Z Ring ◽

Single Point Mutations ◽

Biochemical Analyses ◽

Ftsz Assembly ◽

The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

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An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

Applied and Environmental Microbiology ◽

10.1128/aem.01515-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 3

Author(s):

Sabino Pacheco ◽

Isabel Gómez ◽

Jorge Sánchez ◽

Blanca-Ines García-Gómez ◽

Mario Soberón ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Double Point ◽

Single Point ◽

Three Dimensional ◽

Point Mutations ◽

Salt Bridge ◽

Dimensional Structure ◽

Cry Toxins ◽

Content Type ◽

Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

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Comparison of Predicted Scaffold-Compatible Sequence Variation in the Triple-Hairpin Structure of Human Immunodeficiency Virus Type 1 gp41 with Patient Data

Journal of Virology ◽

10.1128/jvi.76.15.7595-7606.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7595-7606 ◽

Cited By ~ 3

Author(s):

Nathalie Boutonnet ◽

Wouter Janssens ◽

Carlo Boutton ◽

Jean-Luc Verschelde ◽

Leo Heyndrickx ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Sequence Variation ◽

Single Point ◽

Point Mutations ◽

Hairpin Structure ◽

Single Amino Acid ◽

Immunodeficiency Virus

ABSTRACT It has been proposed that the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 (e-gp41), involved in HIV entry into the target cell, exists in at least two conformations, a pre-hairpin intermediate and a fusion-active hairpin structure. To obtain more information on the structure-sequence relationship in e-gp41, we performed in silico a full single-amino-acid substitution analysis, resulting in a Fold Compatible Database (FCD) for each conformation. The FCD contains for each residue position in a given protein a list of values assessing the energetic compatibility (ECO) of each of the 20 natural amino acids at that position. Our results suggest that FCD predictions are in good agreement with the sequence variation observed for well-validated e-gp41 sequences. The data show that at a minECO threshold value of 5 kcal/mol, about 90% of the observed patient sequence variation is encompassed by the FCD predictions. Some inconsistent FCD predictions at N-helix positions packing against residues of the C helix suggest that packing of both peptides may involve some flexibility and may be attributed to an altered orientation of the C-helical domain versus the N-helical region. The permissiveness of sequence variation in the C helices is in agreement with FCD predictions. Comparison of N-core and triple-hairpin FCDs suggests that the N helices may impose more constraints on sequence variation than the C helices. Although the observed sequences of e-gp41 contain many multiple mutations, our method, which is based on single-point mutations, can predict the natural sequence variability of e-gp41 very well.

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