Biology subjects

Guskov, A.

Publications and source records attributed to Guskov, A..

11 recordsLinked to original sources

bioRxiv2025

Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification

Understanding and ultimately predicting the function of proteins is one of the frontiers in structural biology. This will only be possible if it becomes feasible to routinely observe proteins on the fast timescales on which they perform their tasks. Recently, laser flash melting and revitrification experiments have improved the time resolution of cryo-electron microscopy (cryo-EM) to microseconds, rendering it fast enough to observe the domain motions of proteins that are frequently linked to function. However, observations have been limited to a time window of just a few hundred microseconds. Here, we introduce time-resolved cryo-EM experiments based on jet vitrification that combine microsecond resolution with an observation window of up to seconds. We use a short laser pulse to initiate protein dynamics, and as they unfold, vitrify the sample with a jet of a liquid cryogen to arrest the dynamics at that point in time. We demonstrate that our approach affords near-atomic spatial resolution and a time resolution of 21 {micro}s. This allows us to observe the photoinduced dynamics of the light-driven sodium pump ErNaR on the microsecond to millisecond timescale. Our experiments significantly expand the ability of cryo-EM to observe protein dynamics across multiple timescales.

biophysics↗

bioRxiv2025

Determination of the water network surrounding the type I pilus from E. coli by cryo-electron microscopy

Type 1 pili are protein filamentous surface structures of Gram-negative bacteria that mediate adhesion to host and play a crucial role in infection. Here, we report the cryogenic electron microscopy structure of the type 1 pilus from E. coli K-12 comprising 15 subunits of the major protein pilin FimA. The final resolution of EM reconstruction was estimated to be in the range from 2.09 to 2.30 [A], which is higher than that of the previously published structure. This improvement in the resolution enabled us to refine side-chain conformations to reliably determine the distances between the side-chain residues participating in the intersubunit interactions, and determine a network of water molecules surrounding the pilus rod. The analysis revealed that water contributes to intersubunit stabilization both through discrete bridging interactions and through extended hydrogen-bonded clusters, thereby supporting both the rigidity and flexibility of the filament. Comparison with a homologous high-resolution pilus model from enterotoxigenic E. coli showed that nearly all "conserved" water molecules i.e., those that are present at equivalent positions in different subunits of our model occupy also equivalent positions across the two structures, under-scoring their functional relevance. At the same time, sequence-specific differences in hydration patterns were observed. These findings highlight the structural and functional importance of water in pilus architecture and provide a more detailed molecular framework for understanding bacterial adhesion. SynopsisThe improvement in the resolution of the Cryo-EM reconstruction for type I pilus from E. coli made it possible to determine the positions of water molecules surrounding the pilus rod and reveal a more detailed picture of interactions between different subunits of the rod.

molecular biology↗

bioRxiv2025

Unveiling the Molecular Architecture of Candida auris Ribosome

Candida auris is an emerging multidrug-resistant fungal pathogen causing life-threatening invasive candidiasis and bloodstream infections (candidemia), posing significant global health challenges. Despite its importance, the protein translation in pathogenic fungi is poorly characterized. Using cryo-electron microscopy and single-particle reconstruction, we resolved high-resolution structures of the 80S ribosome from C. auris in its vacant state and in complexes with three inhibitors: cycloheximide (CHX), blasticidin-S (BLS), and geneticin (G418). We uncovered a unique substitution of a key nucleotide in the P-site of the small ribosomal subunit (C1160 in C. auris), which may influence ribosome-tRNA interactions and translation fidelity. Comparative analysis of ribosome inhibitor interactions showed that resistance to CHX was observed in only two Candida species examined, while BLS binding displayed no significant differences between C. auris and S. cerevisiae, although C. auris was more sensitive to it. We identified that G418 exhibits promiscuous binding across multiple nonspecific sites, yet its primary interaction site at the decoding center remains highly conserved among Candida species. These findings provide a previously uncharacterized structure of the C. auris ribosome, highlighting novel features that may be leveraged for the development of targeted antifungal therapies to combat multidrug resistance. These insights not only enhance our understanding of ribosomal inhibitor interactions but also suggest potential biomarkers for predicting antifungal susceptibility in clinical applications.

molecular biology↗

bioRxiv2025

Condition-dependent, amorphous protein agglomerates control cytoplasmic rheology

Molecular crowding in the bacterial cytoplasm restricts the diffusion of large molecules, impacting cellular processes. However, how nutrient availability influences cytoplasmic rheology is not well understood. With single-particle tracking in Escherichia coli, we observed a threefold variation in the diffusion of a 40-nm particle across exponential growth conditions. Previously suggested determinants of rheology did not account for this variation; instead, we found a strong anticorrelation between the diffusion coefficient and the abundance of amino acid metabolism proteins, persisting upon genetic perturbations and showing that lower diffusion is associated with increased viscoelasticity. Photoactivated light microscopy revealed that some amino acid metabolism proteins form clusters. Electron microscopy showed that these proteins could form amorphous agglomerates at physiological concentrations in vitro, likely driven by their low intrinsic disorder, high compactness and hydropathy score. These findings show that protein agglomerates regulate cytoplasmic rheology in a condition-dependent manner, suggesting an underappreciated level of cytoplasmic organization. HighlightsO_LIDiffusion of 40-nm particles varies threefold across growth conditions in E. coli C_LIO_LICytoplasmic diffusion inversely correlates with COG-E protein abundance COG-E proteins form agglomerates that increase cytoplasmic viscoelasticity C_LIO_LIProtein compactness and hydrophobicity predict condition-dependent crowding effects C_LI

biophysics↗

bioRxiv2025

Structural basis for no retinal binding in flotillin-associated rhodopsins

Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins were reported to lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins) were identified and suggested to lack the typical retinal binding pocket despite preserving the lysine residue in many members of the group. Here we present cryo-EM structures of paralog FArhodopsin and proteorhodopsin from marine bacteria Pseudothioglobus. The structures revealed pentameric assemblies of both proteins similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Thus, our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of the FArhodopsin clade.

biophysics↗

bioRxiv2024

Structural and biochemical analysis of a B12 super-binder

The gut microbiota is pivotal to human health, playing an important role in nutrient absorption and immune system. A prominent bacterial family in the human gut is Bacteroidetes. Cobalamin (Vitamin B12) is an essential micronutrient for these gut bacteria. Here, we focused on the BtuG family proteins in B. thetaiotaomicron, a model organism for this family, particularly the three homologs BtuG1, BtuG2, and BtuG3, which serve as cobalamin scavengers. Our study aimed to understand the structural and biochemical attributes of these proteins to describe their function in cobalamin acquisition. We solved the crystal structures of all three BtuG homologs bound to different cobalamin forms and the precursor cobinamide, and measured the binding kinetics by the grained coupled interferometry (GCI) technology. Our results reveal high binding affinities, in the low picomolar range underlining their critical role in cobalamin scavenging. Leveraging the high-resolution crystal structures, we successfully designed mutants of these BtuG homologs, resulting in both increased and decreased binding affinities for cobalamin and its precursor. This study furthers our understanding of bacterial adaptation mechanisms in the gut environment, and reveals a family of proteins with unique characteristics toward B12 scavenging, with implications for medical and biotechnological research.

biochemistry↗

bioRxiv2024

Unravelled proteins form blobs during translocation across nanopores

The electroosmotic-driven transport of unravelled proteins across nanopores is an important biological process that is now under investigation for the rapid analysis and sequencing of proteins. For this approach to work, however, it is crucial that the polymer is threaded in single file. Here we found that, contrary to the electrophoretic transport of charged polymers such as DNA, during polypeptide translocation blob-like structures typically form inside nanopores. Comparisons between different nanopore sizes, shapes and surface chemistries showed that under electroosmotic-dominated regimes single-file transport of polypeptides can be achieved using nanopores that simultaneously have an entry and an internal diameter that is smaller than the persistence length of the polymer, have a uniform non-sticky (i.e. non-aromatic) nanopore inner surface, and using moderate translocation velocities.

biophysics↗

bioRxiv2024

CryoRhodopsins: a comprehensive characterization of a new clade of microbial rhodopsins from cold environments

Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.

biophysics↗

bioRxiv2023

A novel subgroup of light-driven sodium pumps with an additional Schiff base counterion

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

biophysics↗

bioRxiv2023

Legionella pneumophila macrophage infectivity potentiator protein appendage domains modulate protein dynamics and inhibitor binding

Macrophage infectivity potentiator (MIP) proteins are widespread in human pathogens including Legionella pneumophila, the causative agent of Legionnaires disease and protozoans such as Trypanosoma cruzi. All MIP proteins contain a FKBP (FK506 binding protein)-like prolyl-cis/trans- isomerase domain that hence presents an attractive drug target. Some MIPs such as the Legionella pneumophila protein (LpMIP) have additional appendage domains of mostly unknown function. In full- length, homodimeric LpMIP, the N-terminal dimerization domain is linked to the FKBP-like domain via a long, free-standing stalk helix. Combining X-ray crystallography, NMR and EPR spectroscopy and SAXS, we elucidated the importance of the stalk helix for protein dynamics and inhibitor binding to the FKBP-like domain and bidirectional crosstalk between the different protein regions. The first comparison of a microbial MIP and a human FKBP in complex with the same synthetic inhibitor was made possible by high-resolution structures of LpMIP with a [4.3.1]-aza-bicyclic sulfonamide and provides a basis for designing pathogen-selective inhibitors. Through stereospecific methylation, the affinity of inhibitors to L. pneumophila and T. cruzi MIP was greatly improved. The resulting X-ray inhibitor-complex structures of LpMIP and TcMIP at 1.49 and 1.34 [A], respectively, provide a starting point for developing potent inhibitors against MIPs from multiple pathogenic microorganisms.

biophysics↗

bioRxiv2020

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid, and asparagine in Gram-positive bacteria. It features two extracytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from L. lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Forster-resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously. HIGHLIGHTSO_LIResolved crystal structure of tandem SBD1-2 of GlnPQ from Lactococcus lactis C_LIO_LIConformational states and ligand binding affinities of individual domains SBD1 and SBD2 are similar to tandem SBD1-2 C_LIO_LINo cooperative effects are seen for different ligands for SBDs in the tandem C_LIO_LIProof of concept experiments show that PIFE-FRET can monitor SBD conformations and protein-protein interaction simultaneously C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/423572v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1fa36f7org.highwire.dtl.DTLVardef@2e973forg.highwire.dtl.DTLVardef@cbae51org.highwire.dtl.DTLVardef@2185cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗