Publications by authors named "Petr V Konarev"

62 Publications

: 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

Limitations of the iterative electron density reconstruction algorithm from solution scattering data.

Nat Methods 2021 03 1;18(3):244-245. Epub 2021 Mar 1.

European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany.

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http://dx.doi.org/10.1038/s41592-021-01082-xDOI Listing
March 2021

The protealysin operon encodes emfourin, a prototype of a novel family of protein metalloprotease inhibitors.

Int J Biol Macromol 2021 Feb 29;169:583-596. Epub 2020 Dec 29.

Institute of Molecular Genetics of National Research Centre "Kurchatov Institute", Moscow, Russia. Electronic address:

Protealysin is a Serratia proteamaculans metalloproteinase of the M4 peptidase family and the prototype of a large group of protealysin-like proteases (PLPs). PLPs are likely involved in bacterial interaction with plants and animals as well as in bacterial pathogenesis. We demonstrated that the PLP genes in bacteria colocalize with the genes of putative conserved proteins. In S. proteamaculans, these two genes form a bicistronic operon. The putative S. proteamaculans protein that we called emfourin (M4in) was expressed in Escherichia coli and characterized. M4in forms a complex with protealysin with a 1:1 stoichiometry and is a potent slow-binding competitive inhibitor of protealysin (K = 52 ± 14 pM); besides, M4in is not secreted from S. proteamaculans constitutively. A comparison of amino acid sequences of M4in and its homologs with those of known inhibitors suggests that M4in is the prototype of a new family of protein inhibitors of proteases.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.12.170DOI Listing
February 2021

Capturing the Conformational Ensemble of the Mixed Folded Polyglutamine Protein Ataxin-3.

Structure 2021 01 15;29(1):70-81.e5. Epub 2020 Oct 15.

The Maurice Wohl Clinical Neuroscience Institute, King's College London, 5 Cutcombe Road, SE5 9RT London, UK; UK Dementia Research Institute at King's College London, SE5 9RT London, UK. Electronic address:

Ataxin-3 is a deubiquitinase involved in protein quality control and other essential cellular functions. It preferentially interacts with polyubiquitin chains of four or more units attached to proteins delivered to the ubiquitin-proteasome system. Ataxin-3 is composed of an N-terminal Josephin domain and a flexible C terminus that contains two or three ubiquitin-interacting motifs (UIMs) and a polyglutamine tract, which, when expanded beyond a threshold, leads to protein aggregation and misfolding and causes spinocerebellar ataxia type 3. The high-resolution structure of the Josephin domain is available, but the structural and dynamical heterogeneity of ataxin-3 has so far hindered the structural description of the full-length protein. Here, we characterize non-expanded and expanded variants of ataxin-3 in terms of conformational ensembles adopted by the proteins in solution by jointly using experimental data from nuclear magnetic resonance and small-angle X-ray scattering with coarse-grained simulations. Our results pave the way to a molecular understanding of polyubiquitin recognition.
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http://dx.doi.org/10.1016/j.str.2020.09.010DOI Listing
January 2021

Author Correction: Octa-repeat domain of the mammalian prion protein mRNA forms stable A-helical hairpin structure rather than G-quadruplexes.

Sci Rep 2020 Mar 4;10(1):4378. Epub 2020 Mar 4.

Institute of Biochemistry and Molecular Biology University of Hamburg, Hamburg, Germany.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-61336-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054425PMC
March 2020

Towards a solution of the inverse X-ray diffraction tomography challenge: theory and iterative algorithm for recovering the 3D displacement field function of Coulomb-type point defects in a crystal.

Acta Crystallogr A Found Adv 2020 Mar 4;76(Pt 2):163-171. Epub 2020 Feb 4.

A.V. Shubnikov Institute of Crystallography of Federal Scientific Research Centre, Leninsky prospekt 59, Moscow 119333, Russian Federation.

The theoretical framework and a joint quasi-Newton-Levenberg-Marquardt-simulated annealing (qNLMSA) algorithm are established to treat an inverse X-ray diffraction tomography (XRDT) problem for recovering the 3D displacement field function f(r - r) = h · u(r - r) due to a Coulomb-type point defect (Ctpd) located at a point r within a crystal [h is the diffraction vector and u(r - r) is the displacement vector]. The joint qNLMSA algorithm operates in a special sequence to optimize the XRDT target function {\cal F}\{ {\cal P} \} in a χ sense in order to recover the function f(r - r) [{\cal P} is the parameter vector that characterizes the 3D function f(r - r) in the algorithm search]. A theoretical framework based on the analytical solution of the Takagi-Taupin equations in the semi-kinematical approach is elaborated. In the case of true 2D imaging patterns (2D-IPs) with low counting statistics (noise-free), the joint qNLMSA algorithm enforces the target function {\cal F} \{ {\cal P} \} to tend towards the global minimum even if the vector {\cal P} in the search is initially chosen rather a long way from the true one.
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http://dx.doi.org/10.1107/S2053273320000145DOI Listing
March 2020

Dodecamers derived from the crystal structure were found in the pre-crystallization solution of the transaminase from the thermophilic bacterium by small-angle X-ray scattering.

J Biomol Struct Dyn 2020 Jul 8;38(10):2939-2944. Epub 2019 Aug 8.

Shubnikov Institute of Crystallography of Federal Scientific Research Centre 'Crystallography and Photonics' of Russian Academy of Sciences, Moscow, Russian Federation.

The pre-crystallization solution of the transaminase from (TaTT) has been studied by small-angle X-ray scattering (SAXS). Regular changes in the oligomeric composition of the protein were observed after the addition of the precipitant. Comparison of the observed oligomers with the crystal structure of TaTT (PDB ID 6GKR) shows that dodecamers may act as building blocks in the growth of transaminase single crystals. Correlating of these results to the similar X-ray studies of other proteins suggests that SAXS may be a valuable tool for searching optimum crystallization conditions. AbbreviationSAXSsmall-angle X-ray scatteringTatransaminaseTaTTtransaminase from PLPpyridoxal-5'-phosphateR-PEAR-(þ)-1-phenylethylamineBCATbranched-chain amino acid aminotransferaseDAATD-aminoacid aminotransferaseR-TAR-amine:pyruvate transaminaseCommunicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2019.1649195DOI Listing
July 2020

Octa-repeat domain of the mammalian prion protein mRNA forms stable A-helical hairpin structure rather than G-quadruplexes.

Sci Rep 2019 02 21;9(1):2465. Epub 2019 Feb 21.

Institute of Biochemistry and Molecular Biology University of Hamburg, Hamburg, Germany.

Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like Creutzfeldt-Jakob disease (CJD) and scrapie. Besides the consensus that spontaneous conversion of normal cellular PrP into misfolded and aggregating PrP is the central event in prion disease, an alternative hypothesis suggests the generation of pathological PrP by rare translational frameshifting events in the octa-repeat domain of the PrP mRNA. Ribosomal frameshifting most commonly relies on a slippery site and an adjacent stable RNA structure to stall translating ribosome. Hence, it is crucial to unravel the secondary structure of the octa-repeat domain of PrP mRNA. Each of the five octa-repeats contains a motif (GGCGGUGGUGGCUGGG) which alone in vitro forms a G-quadruplex. Since the propensity of mRNA to form secondary structure depends on the sequence context, we set to determine the structure of the complete octa-repeat region. We assessed the structure of full-length octa-repeat domain of PrP mRNA using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), circular dichroism (CD) spectroscopy and selective 2'-hydroxyl acylation analysis by primer extension (SHAPE). Our data show that the PrP octa-repeat mRNA forms stable A-helical hairpins with no evidence of G-quadruplex structure even in the presence of G-quadruplex stabilizing agents.
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http://dx.doi.org/10.1038/s41598-019-39213-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384910PMC
February 2019

Pre-crystallization phase formation of thermolysin hexamers in solution close to crystallization conditions.

J Biomol Struct Dyn 2019 08 23;37(12):3058-3064. Epub 2018 Dec 23.

a National Research Centre "Kurchatov Institute" , Moscow , Russian Federation.

Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2018.1507839DOI Listing
August 2019

The Structure of the Pro-domain of Mouse proNGF in Contact with the NGF Domain.

Structure 2019 01 1;27(1):78-89.e3. Epub 2018 Nov 1.

Maurice Wohl Institute, King's College London, 5 Cutcombe Road, London SE5 9RT, UK; Molecular Medicine Department, University of Pavia, Via Carlo Forlanini 6, 27100 Pavia, Italy. Electronic address:

Nerve growth factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation and survival of target neurons. Expressed as a proNGF precursor, NGF is matured by furin-mediated protease cleavage. Increasing evidence suggests that NGF and proNGF have distinct functional roles. While the structure of mature NGF is available, little is known about that of the pro-domain because of its dynamical structural features. We exploited an ad hoc hybrid strategy based on nuclear magnetic resonance and modeling validated by small-angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF. We show that the isolated pro-domain is intrinsically unstructured but forms transient intramolecular contacts with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence. Our data represent an important step toward the structural and functional characterization of the properties of proNGF.
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http://dx.doi.org/10.1016/j.str.2018.09.013DOI Listing
January 2019

3D structure of the natural tetrameric form of human butyrylcholinesterase as revealed by cryoEM, SAXS and MD.

Biochimie 2019 Jan 29;156:196-205. Epub 2018 Oct 29.

National Research Center, Kurchatov Institute, Akademika Kurchatova pl. 1, Moscow, 123182, Russia; Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" Russian Academy of Sciences, Leninsky pr. 59, Moscow, 119333, Russia.

Human plasma butyrylcholinesterase (BChE) is an endogenous bioscavenger that hydrolyzes numerous medicamentous and poisonous esters and scavenges potent organophosphorus nerve agents. BChE is thus a marker for the diagnosis of OP poisoning. It is also considered a therapeutic target against Alzheimer's disease. Although the X-ray structure of a partially deglycosylated monomer of human BChE was solved 15 years ago, all attempts to determine the 3D structure of the natural full-length glycosylated tetrameric human BChE have been unsuccessful so far. Here, a combination of three complementary structural methods-single-particle cryo-electron microscopy, molecular dynamics and small-angle X-ray scattering-were implemented to elucidate the overall structural and spatial organization of the natural tetrameric human plasma BChE. A 7.6 Å cryoEM map clearly shows the major features of the enzyme: a dimer of dimers with a nonplanar monomer arrangement, in which the interconnecting super helix complex PRAD-(WAT)-peptide C-terminal tail is located in the center of the tetramer, nearly perpendicular to its plane, and is plunged deep between the four subunits. Molecular dynamics simulations allowed optimization of the geometry of the molecule and reconstruction of the structural features invisible in the cryoEM density, i.e., glycan chains and glycan interdimer contact areas, as well as intermonomer disulfide bridges at the C-terminal tail. Finally, SAXS data were used to confirm the consistency of the obtained model with the experimental data. The tetramer organization of BChE is unique in that the four subunits are joined at their C-termini through noncovalent contacts with a short polyproline-rich peptide. This tetramer structure could serve as a model for the design of highly stable glycosylated tetramers.
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http://dx.doi.org/10.1016/j.biochi.2018.10.017DOI Listing
January 2019

Direct shape determination of intermediates in evolving macromolecular solutions from small-angle scattering data.

IUCrJ 2018 Jul 30;5(Pt 4):402-409. Epub 2018 May 30.

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

Many important biological processes like amyloid formation, viral assembly can be monitored . Small-angle X-ray scattering (SAXS) is one of the most effective techniques to structurally characterize these processes in solution. For monodisperse systems and some oligomeric mixtures, low-resolution shapes can be determined from the SAXS data, but for evolving systems, such analysis is hampered by the presence of multiple species and no direct reconstruction procedures are available. The authors consider a frequently occurring case where the scattering from the initial and final states of the process are known but there exists a major (unknown) intermediate component. A method is presented to directly reconstruct the low-resolution shape of this transient component together with its volume fractions from multiple scattering patterns recorded from an evolving system. The method is implemented in the computer program freely available to academic users and its effectiveness is illustrated in several synthetic and experimental examples.
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http://dx.doi.org/10.1107/S2052252518005900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038953PMC
July 2018

Optical and Structural Characterization of a Chronic Myeloid Leukemia DNA Biosensor.

ACS Chem Biol 2018 05 23;13(5):1235-1242. Epub 2018 Mar 23.

UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia , Universidade Nova de Lisboa , Campus da Caparica , 2829-516 Caparica , Portugal.

Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on steady-state fluorescence spectroscopy, small-angle X-ray scattering (SAXS), microscale thermophoresis (MST), and differential electrophoretic mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target. We further assessed the structural alterations leading to the specific fluorescence FRET signature by SAXS, MST, and the differential electrophoretic mobility, where the size range observed is consistent with hybridization and formation of a 1:1:1 complex for the probe in the presence of the complementary target and revelator. These results highlight the importance of different techniques to explore conformational DNA changes in solution and its potential to design and characterize molecular biosensors for genetic disease diagnosis.
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http://dx.doi.org/10.1021/acschembio.8b00029DOI Listing
May 2018

The Molecular Bases of the Dual Regulation of Bacterial Iron Sulfur Cluster Biogenesis by CyaY and IscX.

Front Mol Biosci 2017 2;4:97. Epub 2018 Feb 2.

The Wohl Institute, King's College London, London, United Kingdom.

IscX (or YfhJ) is a protein of unknown function which takes part in the iron-sulfur cluster assembly machinery, a highly specialized and essential metabolic pathway. IscX binds to iron with low affinity and interacts with IscS, the desulfurase central to cluster assembly. Previous studies have suggested a competition between IscX and CyaY, the bacterial ortholog of frataxin, for the same binding surface of IscS. This competition could suggest a link between the two proteins with a functional significance. Using a hybrid approach based on nuclear magnetic resonance, small angle scattering and biochemical methods, we show here that IscX is a modulator of the inhibitory properties of CyaY: by competing for the same site on IscS, the presence of IscX rescues the rates of enzymatic cluster formation which are inhibited by CyaY. The effect is stronger at low iron concentrations, whereas it becomes negligible at high iron concentrations. These results strongly suggest the mechanism of the dual regulation of iron sulfur cluster assembly under the control of iron as the effector.
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http://dx.doi.org/10.3389/fmolb.2017.00097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801593PMC
February 2018

A dystroglycan mutation (p.Cys667Phe) associated to muscle-eye-brain disease with multicystic leucodystrophy results in ER-retention of the mutant protein.

Hum Mutat 2018 02 7;39(2):266-280. Epub 2017 Dec 7.

Istituto di Chimica del Riconoscimento Molecolare - CNR c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Roma, Italy.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated α-DG and the transmembrane β-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of α-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of α-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the α-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the β-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of α-DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high-resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small-angle X-ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the β-DG ectodomain overall folding, resulting in disulfide-associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum-retention, underlying dystroglycanopathy.
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http://dx.doi.org/10.1002/humu.23370DOI Listing
February 2018

The effect of the pathological V72I, D109N and T190M missense mutations on the molecular structure of α-dystroglycan.

PLoS One 2017 16;12(10):e0186110. Epub 2017 Oct 16.

Istituto di Cristallografia-CNR, Trieste Outstation, Trieste, Italy.

Dystroglycan (DG) is a highly glycosylated protein complex that links the cytoskeleton with the extracellular matrix, mediating fundamental physiological functions such as mechanical stability of tissues, matrix organization and cell polarity. A crucial role in the glycosylation of the DG α subunit is played by its own N-terminal region that is required by the glycosyltransferase LARGE. Alteration in this O-glycosylation deeply impairs the high affinity binding to other extracellular matrix proteins such as laminins. Recently, three missense mutations in the gene encoding DG, mapped in the α-DG N-terminal region, were found to be responsible for hypoglycosylated states, causing congenital diseases of different severity referred as primary dystroglycanopaties.To gain insight on the molecular basis of these disorders, we investigated the crystallographic and solution structures of these pathological point mutants, namely V72I, D109N and T190M. Small Angle X-ray Scattering analysis reveals that these mutations affect the structures in solution, altering the distribution between compact and more elongated conformations. These results, supported by biochemical and biophysical assays, point to an altered structural flexibility of the mutant α-DG N-terminal region that may have repercussions on its interaction with LARGE and/or other DG-modifying enzymes, eventually reducing their catalytic efficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186110PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643065PMC
October 2017

Block and Gradient Copoly(2-oxazoline) Micelles: Strikingly Different on the Inside.

J Phys Chem Lett 2017 Aug 2;8(16):3800-3804. Epub 2017 Aug 2.

Department of Organic and Macromolecular Chemistry, Ghent University , Krijgslaan 281-S4, 9000 Ghent, Belgium.

Herein, we provide a direct proof for differences in the micellar structure of amphiphilic diblock and gradient copolymers, thereby unambiguously demonstrating the influence of monomer distribution along the polymer chains on the micellization behavior. The internal structure of amphiphilic block and gradient co poly(2-oxazolines) based on the hydrophilic poly(2-methyl-2-oxazoline) (PMeOx) and the hydrophobic poly(2-phenyl-2-oxazoline) (PPhOx) was studied in water and water-ethanol mixtures by small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), static and dynamic light scattering (SLS/DLS), and H NMR spectroscopy. Contrast matching SANS experiments revealed that block copolymers form micelles with a uniform density profile of the core. In contrast to popular assumption, the outer part of the core of the gradient copolymer micelles has a distinctly higher density than the middle of the core. We attribute the latter finding to back-folding of chains resulting from hydrophilic-hydrophobic interactions, leading to a new type of micelles that we refer to as micelles with a "bitterball-core" structure.
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http://dx.doi.org/10.1021/acs.jpclett.7b01588DOI Listing
August 2017

Octamer formation in lysozyme solutions at the initial crystallization stage detected by small-angle neutron scattering.

Acta Crystallogr D Struct Biol 2017 Jul 28;73(Pt 7):591-599. Epub 2017 Jun 28.

Shubnikov Institute of Crystallography, Federal Scientific Research Centre `Crystallography and Photonics', Russian Academy of Sciences, Leninskii pr. 59, Moscow 119333, Russian Federation.

Solutions of lysozyme in heavy water were studied by small-angle neutron scattering (SANS) at concentrations of 40, 20 and 10 mg ml with and without the addition of precipitant, and at temperatures of 10, 20 and 30°C. In addition to the expected protein monomers, dimeric and octameric species were identified in solutions at the maximum concentration and close to the optimal conditions for crystallization. An optimal temperature for octamer formation was identified and both deviation from this temperature and a reduction in protein concentration led to a significant decrease in the volume fractions of octamers detected. In the absence of precipitant, only monomers and a minor fraction of dimers are present in solution.
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http://dx.doi.org/10.1107/S2059798317007422DOI Listing
July 2017

Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction.

Circ Cardiovasc Genet 2016 Oct 13;9(5):426-435. Epub 2016 Sep 13.

Background: High throughput next-generation sequencing techniques have made whole genome sequencing accessible in clinical practice; however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging.

Methods And Results: Here we combine whole genome sequencing with linkage analysis in a 3-generation family affected by cardiomyopathy with features of autosomal dominant left ventricular noncompaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease among the 7 surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterized in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small-angle x ray scattering, and circular dichroism spectroscopy suggest partial unfolding and domain destabilization in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin.

Conclusions: Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left ventricular noncompaction. This expands the spectrum of titin's roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left uninterpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068189PMC
http://dx.doi.org/10.1161/CIRCGENETICS.116.001431DOI Listing
October 2016

Rapid automated superposition of shapes and macromolecular models using spherical harmonics.

J Appl Crystallogr 2016 Jun 16;49(Pt 3):953-960. Epub 2016 May 16.

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

A rapid algorithm to superimpose macromolecular models in Fourier space is proposed and implemented (). The method uses a normalized integrated cross-term of the scattering amplitudes as a proximity measure between two three-dimensional objects. The reciprocal-space algorithm allows for direct matching of heterogeneous objects including high- and low-resolution models represented by atomic coordinates, beads or dummy residue chains as well as electron microscopy density maps and inhomogeneous multi-phase models ( of protein-nucleic acid complexes). Using spherical harmonics for the computation of the amplitudes, the method is up to an order of magnitude faster than the real-space algorithm implemented in by Kozin & Svergun [ (2001 ▸), , 33-41]. The utility of the new method is demonstrated in a number of test cases and compared with the results of . The spherical harmonics algorithm is best suited for low-resolution shape models, . those provided by solution scattering experiments, but also facilitates a rapid cross-validation against structural models obtained by other methods.
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http://dx.doi.org/10.1107/S1600576716005793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886985PMC
June 2016

X-Ray Solution Scattering Study of Four Escherichia coli Enzymes Involved in Stationary-Phase Metabolism.

PLoS One 2016 26;11(5):e0156105. Epub 2016 May 26.

EMBL, Hamburg Outstation, c/o DESY, Hamburg, Germany.

The structural analyses of four metabolic enzymes that maintain and regulate the stationary growth phase of Escherichia coli have been performed primarily drawing on the results obtained from solution small angle X-ray scattering (SAXS) and other structural techniques. The proteins are (i) class I fructose-1,6-bisphosphate aldolase (FbaB); (ii) inorganic pyrophosphatase (PPase); (iii) 5-keto-4-deoxyuronate isomerase (KduI); and (iv) glutamate decarboxylase (GadA). The enzyme FbaB, that until now had an unknown structure, is predicted to fold into a TIM-barrel motif that form globular protomers which SAXS experiments show associate into decameric assemblies. In agreement with previously reported crystal structures, PPase forms hexamers in solution that are similar to the previously reported X-ray crystal structure. Both KduI and GadA that are responsible for carbohydrate (pectin) metabolism and acid stress responses, respectively, form polydisperse mixtures consisting of different oligomeric states. Overall the SAXS experiments yield additional insights into shape and organization of these metabolic enzymes and further demonstrate the utility of hybrid methods, i.e., solution SAXS combined with X-ray crystallography, bioinformatics and predictive 3D-structural modeling, as tools to enrich structural studies. The results highlight the structural complexity that the protein components of metabolic networks may adopt which cannot be fully captured using individual structural biology techniques.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156105PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881948PMC
July 2017

Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure.

Biochemistry 2016 Jan 14;55(3):560-74. Epub 2016 Jan 14.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences , Magyar tudósok krt. 2., H-1117 Budapest, Hungary.

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure.
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http://dx.doi.org/10.1021/acs.biochem.5b00839DOI Listing
January 2016

A posteriori determination of the useful data range for small-angle scattering experiments on dilute monodisperse systems.

IUCrJ 2015 May 21;2(Pt 3):352-60. Epub 2015 Apr 21.

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

Small-angle X-ray and neutron scattering (SAXS and SANS) experiments on solutions provide rapidly decaying scattering curves, often with a poor signal-to-noise ratio, especially at higher angles. On modern instruments, the noise is partially compensated for by oversampling, thanks to the fact that the angular increment in the data is small compared with that needed to describe adequately the local behaviour and features of the scattering curve. Given a (noisy) experimental data set, an important question arises as to which part of the data still contains useful information and should be taken into account for the interpretation and model building. Here, it is demonstrated that, for monodisperse systems, the useful experimental data range is defined by the number of meaningful Shannon channels that can be determined from the data set. An algorithm to determine this number and thus the data range is developed, and it is tested on a number of simulated data sets with various noise levels and with different degrees of oversampling, corresponding to typical SAXS/SANS experiments. The method is implemented in a computer program and examples of its application to analyse the experimental data recorded under various conditions are presented. The program can be employed to discard experimental data containing no useful information in automated pipelines, in modelling procedures, and for data deposition or publication. The software is freely accessible to academic users.
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http://dx.doi.org/10.1107/S2052252515005163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420545PMC
May 2015

Coat Protein-Dependent Behavior of Poly(ethylene glycol) Tails in Iron Oxide Core Virus-like Nanoparticles.

ACS Appl Mater Interfaces 2015 Jun 29;7(22):12089-98. Epub 2015 May 29.

†Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States.

Here we explore the formation of virus-like nanoparticles (VNPs) utilizing 22-24 nm iron oxide nanoparticles (NPs) as cores and proteins derived from viral capsids of brome mosaic virus (BMV) or hepatitis B virus (HBV) as shells. To accomplish that, hydrophobic FeO/Fe3O4 NPs prepared by thermal decomposition of iron oleate were coated with poly(maleic acid-alt-octadecene) modified with poly(ethylene glycol) (PEG) tails of different lengths and grafting densities. MRI studies show high r2/r1 relaxivity ratios of these NPs that are practically independent of the polymer coating type. The versatility and flexibility of the viral capsid protein are on display as they readily form shells that exceed their native size. The location of the long PEG tails upon shell formation was investigated by electron microscopy and small-angle X-ray scattering. PEG tails were located differently in the BMV and HBV VNPs, with the BMV VNPs preferentially entrapping the tails in the interior and the HBV VNPs allowing the tails to extend through the capsid, which highlights the differences between intersubunit interactions in these two icosahedral viruses. The robustness of the assembly reaction and the protruding PEG tails, potentially useful in modulating the immune response, make the systems introduced here a promising platform for biomedical applications.
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http://dx.doi.org/10.1021/acsami.5b02278DOI Listing
June 2015

Allosteric regulation of deubiquitylase activity through ubiquitination.

Front Mol Biosci 2015 5;2. Epub 2015 Feb 5.

Department of Clinical Neuroscience, King's College London London, UK.

Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.
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http://dx.doi.org/10.3389/fmolb.2015.00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428445PMC
May 2015

Structural insights into Ca2+-calmodulin regulation of Plectin 1a-integrin β4 interaction in hemidesmosomes.

Structure 2015 Mar 19;23(3):558-570. Epub 2015 Feb 19.

Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter (VBC), Campus Vienna Biocenter 5, A-1030 Vienna, Austria; Department of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia. Electronic address:

The mechanical stability of epithelial cells, which protect organisms from harmful external factors, is maintained by hemidesmosomes via the interaction between plectin 1a (P1a) and integrin α6β4. Binding of calcium-calmodulin (Ca(2+)-CaM) to P1a together with phosphorylation of integrin β4 disrupts this complex, resulting in disassembly of hemidesmosomes. We present structures of the P1a actin binding domain either in complex with the N-ter lobe of Ca(2+)-CaM or with the first pair of integrin β4 fibronectin domains. Ca(2+)-CaM binds to the N-ter isoform-specific tail of P1a in a unique manner, via its N-ter lobe in an extended conformation. Structural, cell biology, and biochemical studies suggest the following model: binding of Ca(2+)-CaM to an intrinsically disordered N-ter segment of plectin converts it to an α helix, which repositions calmodulin to displace integrin β4 by steric repulsion. This model could serve as a blueprint for studies aimed at understanding how Ca(2+)-CaM or EF-hand motifs regulate F-actin-based cytoskeleton.
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http://dx.doi.org/10.1016/j.str.2015.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353693PMC
March 2015

The conundrum of the high-affinity NGF binding site formation unveiled?

Biophys J 2015 Feb;108(3):687-97

European Brain Research Institute, Roma, Italy; Scuola Normale Superiore, Pisa, Italy. Electronic address:

The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and p75(NTR). Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary TrkA/NGF/p75(NTR) complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a TrkA/NGF/p75(NTR) ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of p75(NTR) with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and p75(NTR)/NGF complexes.
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http://dx.doi.org/10.1016/j.bpj.2014.11.3485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317559PMC
February 2015

Glutamate 270 plays an essential role in K(+)-activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase.

FEBS Lett 2015 Jan 10;589(2):240-5. Epub 2014 Dec 10.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary. Electronic address:

The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K(+) compared to the wild-type enzyme. A 3-4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K(+). SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K(+) into close proximity of the nicotinamide ring of NAD(+) and the electron-withdrawing effect of K(+) may help to polarise the aromatic ring in order to aid the hydride-transfer.
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http://dx.doi.org/10.1016/j.febslet.2014.12.005DOI Listing
January 2015

The structure and regulation of human muscle α-actinin.

Cell 2014 Dec 26;159(6):1447-60. Epub 2014 Nov 26.

Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030 Vienna, Austria; Department of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, 1000 Ljubljana, Slovenia. Electronic address:

The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.
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http://dx.doi.org/10.1016/j.cell.2014.10.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259493PMC
December 2014
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