Biology subjects

Nikolaev, A.

Publications and source records attributed to Nikolaev, A..

12 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↗

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

Different behaviourally relevant stimuli evoke different forms of adaptation in the olfactory system

Sensory neurons are continuously exposed to a large diversity of stimuli and adjust their response according to the recent stimulation history, in a process called adaptation. Recent studies have demonstrated that many sensory neurons not only depress (decrease their response to repetitive sensory stimulation) but some neurons exhibit facilitation: a small initial response followed by increase in response amplitude. Adaptation has been mainly studied with neutral stimuli and it is not known whether different behaviourally relevant stimuli evoke adaptation with similar or different properties. Here we used ethologically relevant stimuli to study adaptation in the zebrafish olfactory system. We found that repetitive presentation of food odour caused a variety of adaptations ranging from very strong depression to facilitation, but depression was the predominant dynamic. On the other hand, a different behaviourally-relevant stimulus, alarm substance, evoked much stronger facilitation and also decreased the amplitude of responses to food presentation. Other sensory modalities, aversive mechanosensory and attractive visual stimuli, also caused adaptation with different properties in the same brain area. Different forms of adaptation may therefore be used for processing sensory stimuli evoking different behavioural reactions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/612693v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1cd5c18org.highwire.dtl.DTLVardef@1592ea1org.highwire.dtl.DTLVardef@4841corg.highwire.dtl.DTLVardef@7d88d9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsFood odour evokes a diversity of adaptations ranging from extreme depression to facilitation Alarm substance evokes much stronger facilitation than food odour Alarm substance inhibits responses to food odour Mechanosensory stimulation evokes both depression and facilitation Visual stimuli evoke responses in the olfactory bulbs that strongly and quickly depress

neuroscience↗

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↗

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↗

bioRxiv2020

An open-source experimental framework for automation of cell biology experiments

Modern data analysis methods, such as optimisation algorithms or machine and deep learning, have been successfully applied to a number of biological, biotechnological and medical questions. For these methods to be efficient, a large number of high quality experiments need to be conducted, which requires a high degree of automation. Here we report an open-source hardware that allows for automatic high-throughput generation of large amounts of cell biology data. The hardware consists of an automatic XY-stage for moving a multiwell plate containing growing cells; a perfusion manifold allowing application of up to 8 different solutions; and a small epifluorescent microscope. It is extremely cheap (approximately {pound}400 without and {pound}2500 with a fluorescent microscope) and is easily customizable for individual experimental needs. We demonstrate the usability of this platform with high-throughput Ca2+ imaging and large-scale labelling experiments. Key points- We present an open source framework for automation of cell biology experiments - The framework consists of an XY platform, application of up to 8 solutions and a small epifluorescent microscope with autofocusing - Very cheap ({pound}400 without a fluorescent microscope and {pound}2500 with a fluorescent microscope), customisable, - Can be used in a variety of biological applications such as imaging of fluorescent reporters, optimisation of treatment conditions and fluorescent labelling

cell biology↗

bioRxiv2019

Co-ordinated Ras and Rac activity shapes macropinocytic cups and enables phagocytosis of geometrically diverse bacteria

Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialised cup shaped protrusions. To effectively capture and internalise their targets, these cups are organised into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac and phospholipid interactions to localise at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, RGBARG shapes the protrusion by driving Rac activation at the rim whilst suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of co-ordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.

cell biology↗

bioRxiv2018

Ca2+-activated K+ channels reduce network excitability, improving adaptability and energetics for transmitting and perceiving sensory information

Ca2+-activated K+ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modelling, we discover how visual information transfer in mutants lacking the BK channel (dSlo-), SK channel (dSK-) or both (dSK-;;dSlo-) is shaped in the Drosophila R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K+ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and distort the mutants motion perception, determining the true price and limits of homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca2+-activated K+ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.\n\nHighlightsO_LIHomeostatic plasticity in Drosophila dSlo-, dSK- and dSK-;;dSlo- null-mutants retains R1-R6 photoreceptors light information sampling while reducing other K+-conductances\nC_LIO_LIAs mutant R-LMC-R circuits rebalance synaptic loads homeostatically, R1-R6s become more depolarized, with dSK- and dSK-;;dSlo- responding faster and dSlo- slower, whilst LMC outputs oscillate, with dSK- responding faster and dSK-;;dSlo- and dSlo- slower than wild-type\nC_LIO_LIHomeostatic compensation in the mutant circuits impedes adaptation, increases the energy cost of visual information and distorts optomotor behavior\nC_LIO_LIHence, Ca2+-activated K+ channels improve adaptability and energetics for transmitting and perceiving sensory information\nC_LI

neuroscience↗