Biology subjects

Gushchin, I.

Publications and source records attributed to Gushchin, I..

17 recordsLinked to original sources

bioRxiv2026

Structure of a dodecameric double-ferritin-fold protein from an Asgard archaeon

Ferritins are ubiquitous iron homeostasis proteins found across the tree of life that form conserved 24-subunit cages with octahedral (4-3-2) symmetry. New types of ferritins and ferritin-like proteins are being continuously discovered, such as mini-bacterioferritins, which form smaller shells of 12 subunits, and double-ferritin-fold proteins, which act as ferroxidases but do not form shells. Here, we describe double-ferritin-fold proteins from Asgard archaea, dubbed dFTNs, and determine Cryo-EM structure of a representative from Candidatus Heimdallarchaeum endolithica. The protein forms a dodecameric shell with tetrahedral (2-3) symmetry. N-terminal (NTD) and C-terminal (CTD) domains are bridged by an ordered linker and are related by two-fold rotational pseudosymmetry. C-terminal -helix (helix E) that forms the four-fold channel in classic ferritins is repositioned to be the helix 2 out of 5 ferritin domain -helices in dFTN, with two such helices from NTD and two helices from CTD forming a pseudo-four-fold symmetry structural element. Four three-fold channels are formed by NTDs, and four other such channels are formed by CTDs. The overall arrangement of dFTN ferritin domains is similar to that of protomers in classic ferritin shells. Altogether, our findings expand the range of known ferritin family proteins and provide insight into Asgard archaea iron metabolism.

biophysics↗

bioRxiv2026

CARS: A General Force Field for Carotenoids

Carotenoids are structurally diverse isoprenoid pigments that play central roles in photosynthesis, photoprotection, membrane organization, and cellular signaling. Despite their biological and technological importance, atomistic simulations of carotenoids remain limited by the lack of a transferable force field spanning the chemical diversity of naturally occurring compounds, including glycosylated and acylated derivatives. Here we present CARS (CARotenoidS), a transferable force field for carotenoids that integrates seamlessly with the AMBER family of biomolecular force fields. Parameters were systematically optimized against 22 957 r2SCAN-3c reference energies for 25 representative molecular fragments, yielding an accurate description of polyene conformational energetics, ring rotations, and molecular geometries. Across diverse validation sets, CARS substantially outperforms GAFF2 and provides improved agreement with quantum-chemical reference data for glycosylated and acylated carotenoids. For zeaxanthin, CARS also surpasses OPLS-AA, CGenFF, and previously published carotenoid-specific parameters set in reproducing conformational energetics and structural properties. Two complementary parameter sets are provided: CARS for glycosylated and non-lipidated carotenoids, and CARS+Lipid21 for carotenoids containing saturated or monounsaturated lipid chains. By providing the first unified and transferable parameterization covering the structural diversity of natural carotenoids while remaining fully compatible with established AMBER force fields, CARS removes the need for molecule-specific reparameterization and enables reliable molecular simulations of carotenoids in proteins, membranes, and other complex biological assemblies.

bioinformatics↗

bioRxiv2026

Influence of molecular representation and charge on protein-ligand structural predictions by popular co-folding methods

Recently developed deep learning-based tools can effectively generate structural models of complexes of proteins and non-proteinaceous compounds. While some of their predictive capabilities are truly exciting, others remain to be thoroughly tested. Here, we probe whether the ligand input format (Chemical Component Dictionary, CCD, or Simplified Molecular Input Line Entry System, SMILES) and charge (which depends on protonation) will affect the results of the predictions by four popular algorithms: AlphaFold 3, Boltz-2, Chai-1, and Protenix-v1. We chose methylamine and acetic acid as two of the simplest titratable chemicals that are omnipresent in proteins as amino and carboxy moieties, and are consequently ubiquitous in the Protein Data Bank models that are most commonly used for training. Unexpectedly, we found that for both molecules, in many cases the input format affected the prediction results, and did it much stronger compared to protonation, whereas changes in the formally specified charge of the molecules did not lead to changes in binding expected from experiments. We conclude that (i) ensuring identical results irrespective of input formats and (ii) inclusion of protonation-related steps into training and prediction pipelines are the two available paths for improvement of protein-ligand structure prediction algorithms.

bioinformatics↗

bioRxiv2025

Structural and Functional Characterization of Encapsulin-Targeted Double Ferritin Fold Ferroxidases

Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four -helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/676036v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@72e6b0org.highwire.dtl.DTLVardef@18752fforg.highwire.dtl.DTLVardef@15a02e7org.highwire.dtl.DTLVardef@74cd02_HPS_FORMAT_FIGEXP M_FIG C_FIG A family of double ferritin-like proteins (DFLPs) composed of two four-helical domains is described and investigated. DFLPs are shown to form homodimers, bind heme, possess diiron sites and display ferroxidase activity. Some DFLPs are shown to be targeted to encapsulin shells as core or secondary cargo, thus representing a new type of iron metabolism systems.

biochemistry↗

bioRxiv2025

Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments.

biochemistry↗

bioRxiv2024

Engineering of soluble bacteriorhodopsin

Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein. Here, we report engineering of soluble analogues of bacteriorhodopsin, NeuroBRs, which bind retinal and photocycle under illumination. We also report the crystallographic structure of NeuroBR_A, determined at anisotropic resolution reaching 1.76 [A], that reveals a conserved chromophore binding pocket and tertiary structure. Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins.

bioengineering↗

bioRxiv2024

High-throughput algorithm predicts F-Type ATP synthase rotor ring stoichiometries of 8 to 27 protomers

ATP synthases are large enzymes present in every living cell. They consist of a transmembrane and a soluble domain, each comprising multiple subunits. The transmembrane part contains an oligomeric rotor ring (c-ring), whose stoichiometry defines the ratio between the number of synthesized ATP molecules and the number of ions transported through the membrane. Currently, c-rings of F-Type ATP synthases consisting of 8 to 17 (except 16) subunits have been experimentally demonstrated. Here, we present an easy-to-use high-throughput computational approach based on AlphaFold that allows us to estimate the stoichiometry of all homooligomeric c-rings, whose sequences are present in genomic databases. We validate the approach on the available experimental data, obtaining the correlation as high as 0.94 for the reference data set, and use it to predict the existence of c-rings with stoichiometry varying from 8 to 27. We then conduct molecular dynamics simulations of two c-rings with stoichiometry above 17 to corroborate the machine learning-based predictions. Our work strongly suggests existence of rotor rings with previously undescribed high stoichiometry in natural organisms and highlights the utility of AlphaFold-based approaches for studying homooligomeric proteins.

bioinformatics↗

bioRxiv2023

Reengineering of a flavin-binding fluorescent protein using ProteinMPNN

Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user-defined backbone structure. In this short report, we test whether ProteinMPNN can be used to reengineer a flavin-binding fluorescent protein, CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN), while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36-48 out of the remaining 86 amino acids. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand-binding capabilities.

bioengineering↗

bioRxiv2023

Two distinct mechanisms of flavoprotein spectral tuning revealed by low-temperature and time-dependent spectroscopy

Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine X-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color tuning mechanisms of flavin-binding proteins and pave the way for engineering of new variants with improved optical properties. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/549366v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1403d8eorg.highwire.dtl.DTLVardef@894bd1org.highwire.dtl.DTLVardef@1d089b1org.highwire.dtl.DTLVardef@1ffd3c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

luxA gene from Enhygromyxa salina encodes a functional homodimeric luciferase

Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is catalyzed by the subunit LuxA, while LuxB is inactive. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product of the luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes both in vivo in Escherichia coli and in vitro. Overall, EsLuxA is less bright compared with luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 [A] reveals a classical monooxygenase fold, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a {beta}-hairpin or a coil in Vibrio harveyi LuxA, form -helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for discovery of new luciferases that have an advantage of being encoded by a single gene.

biochemistry↗

bioRxiv2022

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Flavin-binding fluorescent proteins (FbFPs) are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of twenty-two finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans (CagFbFP). We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of FbFPs in fluorescence microscopy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/519645v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@786ee9org.highwire.dtl.DTLVardef@b99199org.highwire.dtl.DTLVardef@105e7aborg.highwire.dtl.DTLVardef@acbd50_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PR) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 [A] resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric subpocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, the new S1P5-inverse agonist structure sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR-family.

biophysics↗

bioRxiv2021

Structure and dynamics of the SARS-CoV-2 envelope protein monomer

Coronaviruses, especially SARS-CoV-2, present an ongoing threat for human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic -helical transmembrane domain (TMD) and amphiphilic -helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43 and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane. This may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate budding of the viral particles. The presented results may be helpful for better understanding of the function of coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.

biophysics↗

bioRxiv2021

Insights into the mechanisms of LOV domain color tuning from a set of high-resolution X-ray structures

Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of [~]440 to [~]450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine amino acid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced by acidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 [A] for Q148D to 1.63 [A] for Q148R. Whereas in some of the variants, the amino acid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged amino acids and pave the way for rational design of color-shifted flavin based fluorescent proteins.

biophysics↗

bioRxiv2021

Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Determining the molecular origin of spectral tuning in photoactive biological systems is instrumental for understanding their function. Spectral-tuning efforts for flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, are limited by their dependency on protein-bound flavins, whose structure and hence electronic properties, cannot be altered by mutation. To address those shortcomings, we here present the photophysical, computational and structural characterization of structurally uncharacterized blue-shifted FbFPs, carrying a previously described lysine substitution within their flavin-binding pocket. X-ray structures reveal displacement of the lysine away from the chromophore and opening up of the structure as cause for the blue shift. Site-saturation mutagenesis and high-throughput screening, yielded a red-shifted variant, in which the lysine side chain of the blue-shifted variant is stabilized in close distance to the flavin by a secondary mutation, mechanistically accounting for the red shift. Thus, a single secondary mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools that should find application in life sciences.

biophysics↗

bioRxiv2020

Molecular mechanism of light-driven sodium pumping

Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics.

biophysics↗

bioRxiv2019

High Resolution Structural Insights into Heliorhodopsin Family

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 [A] showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (active site), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the active site which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes.

biophysics↗