Publications by authors named "Andrey Gruzinov"

9 Publications

  • Page 1 of 1

Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations.

Int J Mol Sci 2021 Jun 3;22(11). Epub 2021 Jun 3.

Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" of Russian Academy of Sciences", Leninskiy Prospect, 59, 119333 Moscow, Russia.

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.
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http://dx.doi.org/10.3390/ijms22116056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199988PMC
June 2021

Molecular model of a sensor of two-component signaling system.

Sci Rep 2021 May 24;11(1):10774. Epub 2021 May 24.

Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141700, Dolgoprudny, Russia.

Two-component systems (TCS) are widespread signaling systems present in all domains of life. TCS typically consist of a signal receptor/transducer and a response regulator. The receptors (histidine kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure. Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit. However, much less is known about photoreceptors. Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities. We then fit an atomistic model of the whole complex into the SAS data. The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected.
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http://dx.doi.org/10.1038/s41598-021-89613-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144572PMC
May 2021

Anomalous SAXS at P12 beamline EMBL Hamburg: instrumentation and applications.

J Synchrotron Radiat 2021 May 14;28(Pt 3):812-823. Epub 2021 Apr 14.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Small-angle X-ray scattering (SAXS) is an established method for studying nanostructured systems and in particular biological macromolecules in solution. To obtain element-specific information about the sample, anomalous SAXS (ASAXS) exploits changes of the scattering properties of selected atoms when the energy of the incident X-rays is close to the binding energy of their electrons. While ASAXS is widely applied to condensed matter and inorganic systems, its use for biological macromolecules is challenging because of the weak anomalous effect. Biological objects are often only available in small quantities and are prone to radiation damage, which makes biological ASAXS measurements very challenging. The BioSAXS beamline P12 operated by the European Molecular Biology Laboratory (EMBL) at the PETRA III storage ring (DESY, Hamburg) is dedicated to studies of weakly scattering objects. Here, recent developments at P12 allowing for ASAXS measurements are presented. The beamline control, data acquisition and data reduction pipeline of the beamline were adapted to conduct ASAXS experiments. Modelling tools were developed to compute ASAXS patterns from atomic models, which can be used to analyze the data and to help designing appropriate data collection strategies. These developments are illustrated with ASAXS experiments on different model systems performed at the P12 beamline.
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http://dx.doi.org/10.1107/S1600577521003404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127372PMC
May 2021

: expanded functionality and new tools for small-angle scattering data analysis.

J Appl Crystallogr 2021 Feb 1;54(Pt 1):343-355. Epub 2021 Feb 1.

European Molecular Biology Laboratory, Hamburg Site, Notkestrasse 85, Building 25 A, Hamburg, 22607, Germany.

The software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the package are described. They include , for simulating isotropic 2D scattering patterns; , to perform operations on 2D images and masks; , a method for variance estimation of structural invariants through parametric resampling; , which computes the pair distance distribution function by a direct Fourier transform of the scattering data; , to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in for Bayesian consensus-based concentration-independent molecular weight estimation; , an shape analysis method that optimizes the search model directly against the scattering data; , an application to set up the initial search volume for multiphase modelling of membrane proteins; , to perform quasi-atomistic modelling of liposomes with elliptical shapes; , which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; , which reconstructs the shape of an unknown intermediate in an evolving system; and and , for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the graphical interface to Qt5, updating - a plugin to run a subset of tools - to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future releases. All these features are implemented in , freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.
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http://dx.doi.org/10.1107/S1600576720013412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941305PMC
February 2021

Restoring structural parameters of lipid mixtures from small-angle X-ray scattering data.

J Appl Crystallogr 2021 Feb 1;54(Pt 1):169-179. Epub 2021 Feb 1.

Hamburg Outstation, European Molecular Biology Laboratory, Notkestrasse 85, Hamburg, 22607, Germany.

Small-angle X-ray scattering (SAXS) is widely utilized to study soluble macromolecules, including those embedded into lipid carriers and delivery systems such as surfactant micelles, phospho-lipid vesicles and bilayered nanodiscs. To adequately describe the scattering from such systems, one needs to account for both the form factor (overall structure) and long-range-order Bragg reflections emerging from the organization of bilayers, which is a non-trivial task. Presently existing methods separate the analysis of lipid mixtures into distinct procedures using form-factor fitting and the fitting of the Bragg peak regions. This article describes a general approach for the computation and analysis of SAXS data from lipid mixtures over the entire angular range of an experiment. The approach allows one to restore the electron density of a lipid bilayer and simultaneously recover the corresponding size distribution and multilamellar organization of the vesicles. The method is implemented in a computer program, , and its performance is demonstrated on an aqueous solution of layered lipid vesicles undergoing an extrusion process. The approach is expected to be useful for the analysis of various types of lipid-based systems, for the characterization of interactions between target drug molecules and potential carrier/delivery systems.
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http://dx.doi.org/10.1107/S1600576720015368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941313PMC
February 2021

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.

Nat Commun 2020 11 4;11(1):5588. Epub 2020 Nov 4.

Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Notkestrasse 85, D-22607, Hamburg, Germany.

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.
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http://dx.doi.org/10.1038/s41467-020-19204-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642358PMC
November 2020

Molecular Mechanisms of the Interactions of -(2-Hydroxypropyl)methacrylamide Copolymers Designed for Cancer Therapy with Blood Plasma Proteins.

Pharmaceutics 2020 Jan 28;12(2). Epub 2020 Jan 28.

Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.

The binding of plasma proteins to a drug carrier alters the circulation of nanoparticles (NPs) in the bloodstream, and, as a consequence, the anticancer efficiency of the entire nanoparticle drug delivery system. We investigate the possible interaction and the interaction mechanism of a polymeric drug delivery system based on -(2-hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) with the most abundant proteins in human blood plasma-namely, human serum albumin (HSA), immunoglobulin G (IgG), fibrinogen (Fbg), and apolipoprotein (Apo) E4 and A1-using a combination of small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), and nuclear magnetic resonance (NMR). Through rigorous investigation, we present evidence of weak interactions between proteins and polymeric nanomedicine. Such interactions do not result in the formation of the protein corona and do not affect the efficiency of the drug delivery.
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http://dx.doi.org/10.3390/pharmaceutics12020106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076460PMC
January 2020

Smaller capillaries improve the small-angle X-ray scattering signal and sample consumption for biomacromolecular solutions.

J Synchrotron Radiat 2018 Jul 26;25(Pt 4):1113-1122. Epub 2018 Jun 26.

European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Radiation damage by intense X-ray beams at modern synchrotron facilities is one of the major complications for biological small-angle X-ray scattering (SAXS) investigations of macromolecules in solution. To limit the damage, samples are typically measured under a laminar flow through a cell (typically a capillary) such that fresh solution is continuously exposed to the beam during measurement. The diameter of the capillary that optimizes the scattering-to-absorption ratio at a given X-ray wavelength can be calculated a priori based on fundamental physical properties. However, these well established scattering and absorption principles do not take into account the radiation susceptibility of the sample or the often very limited amounts of precious biological material available for an experiment. Here it is shown that, for biological solution SAXS, capillaries with smaller diameters than those calculated from simple scattering/absorption criteria allow for a better utilization of the available volumes of radiation-sensitive samples. This is demonstrated by comparing two capillary diameters d (d = 1.7 mm, close to optimal for 10 keV; and d = 0.9 mm, which is nominally sub-optimal) applied to study different protein solutions at various flow rates. The use of the smaller capillaries ultimately allows one to collect higher-quality SAXS data from the limited amounts of purified biological macromolecules.
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http://dx.doi.org/10.1107/S1600577518007907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038601PMC
July 2018

Thermally induced conformational changes and protein-protein interactions of bovine serum albumin in aqueous solution under different pH and ionic strengths as revealed by SAXS measurements.

Phys Chem Chem Phys 2017 Jul;19(26):17143-17155

Università Politecnica delle Marche, Dipartimento di Scienze della Vita e dell'Ambiente, Ancona, Italy.

Thermal-induced conformational changes and protein-protein interactions of bovine serum albumin (BSA) in aqueous solution are assessed by small angle X-ray scattering (SAXS) at two pH values (7.4 and 9.0) and two ionic strengths (0.1 and 0.5). We demonstrate that Guinier analysis in two ranges of the modulus of the scattering vector allows protein melting and aggregation to be monitored simultaneously, thus providing insights into the mechanism of thermal-induced BSA aggregation. Results of the analysis suggest that at room temperature monomeric and dimeric BSA fractions are present in solution. For low concentrations (<10 mg mL) the monomeric to dimeric fraction ratio is close to 6, the same value we obtained independently in size-exclusion chromatography experiments. For elevated concentrations (20 mg mL and 40 mg mL) a decrease in the dimer fraction occurs. Following heating, dimer formation is observed prior to protein melting, while no higher order aggregates are observed in the 20-60 °C temperature range. In the vicinity of the BSA melting point, higher order aggregates appear and protein molecules exhibit an aggregation burst. Higher ionic strength makes the described effects more pronounced - dimer formation increases at lower temperatures, presumably due to partial screening of electrostatic interactions between protein molecules. Moreover, the melting temperature shifts to higher values upon increasing the protein concentration and pH, indicating that repulsive interactions stabilize the protein structure. The suggested model was verified by the assessment of parameters of protein-protein interaction potentials based on DLVO theory using the global fitting procedure.
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http://dx.doi.org/10.1039/c6cp08809kDOI Listing
July 2017
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