Wiktor Koźmiński's NMR group

Biological and Chemical Research Centre, University of Warsaw

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Wiktor Koźmiński's NMR Group

Open Positions

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Project: ‘New tools and applications of NMR spectroscopy beyond resolution limitation.’

Project coordinator: prof. Wiktor Koźmiński.
Project duration: 2016 - 2021.

 

Positions for MSc students and Postdoc available.

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The aim of the project is an expansion of capabilities of the high-resolution nuclear magnetic resonance (NMR) spectroscopy which is a fundamental tool of modern structural biology. The structure and dynamics of proteins will be studied using new spectral parameters, such as cross-correlated relaxation rates. The research conducted in the frames of the project will make use of multidimensional NMR spectroscopy of isotopically enriched samples (13C, 15N, 2H) of proteins, both of folded and disordered nature. In addition, high hydrostatic pressure NMR will be employed to study conformational equilibria and dynamics of investigated proteins. Exceptionally high-resolution of 4 and 5 dimensional spectra will be achieved thanks to non-uniform sampling and advanced processing tools.

All positions are available from June, 2016. Candidate for MSc position shoud hold BSc preferably in chemistry, physics, biology or computer-science. All stipends are funded from NCN MAESTRO grant.

Postdoc candidates are asked to directly contact project coordinator.

More info: prof. Wiktor Koźmiński, This e-mail address is being protected from spambots. You need JavaScript enabled to view it
Application deadline: ongoing recruitment.

 

New Article in Scientific Reports

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Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

Tomasz Kochańczyk, Michał Nowakowski, Dominika Wojewska, Andrzej Ejchart, Wiktor Koźmiński, Artur Krężel


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The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

 

New Article in Journal of Biomolecular NMR

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Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein

Szymon Żerko, Piotr Byrski, Paweł Włodarczyk-Pruszyński, Michał Górka, Karin Ledolter, Eliezer Masliah, Robert Konrat, Wiktor Koźmiński


5Ds

New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

 

New Article in Journal of Biomolecular NMR

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Amino acid recognition for automatic resonance assignment of intrinsically disordered proteins

Alessandro Piai, Leonardo Gonnelli, Isabella C. Felli, Roberta Pierattelli, Krzysztof Kazimierczuk, Katarzyna Grudziąż, Wiktor Koźmiński, Anna Zawadzka-Kazimierczuk


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Resonance assignment is a prerequisite for almost any NMR-based study of proteins. It can be very challenging in some cases, however, due to the nature of the protein under investigation. This is the case with intrinsically disordered proteins, for example, whose NMR spectra suffer from low chemical shifts dispersion and generally low resolution. For these systems, sequence specific assignment is highly time-consuming, so the prospect of using automatic strategies for their assignment is very attractive. In this article we present a new version of the automatic assignment program TSAR dedicated to intrinsically disordered proteins. In particular, we demonstrate how the automatic procedure can be improved by incorporating methods for amino acid recognition and information on chemical shifts in selected amino acids. The approach was tested in silico on 16 disordered proteins and experimentally on α-synuclein, with remarkably good results.

 

New Article in Journal of Biomolecular NMR

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Nuclear overhauser spectroscopy of chiral CHD methylene groups

Rafal Augustyniak, Jan Stanek,  Henri Colaux, Geoffrey Bodenhausen, Wiktor Koźmiński, Torsten Hermann, Fabien Ferrage


Janek JBIO

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.

 

A to B Transition

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'A' state

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Transition state

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'B' state

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New Article in Journal of Biological Chemistry

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Biochemical and structural characterization of the interaction between the Siderocalin NGAL/LCN2 and the N-terminal domain of its endocytic receptor SLC22A17

Ana-Isabel Cabedo Martinez, Katharina Weinhaupl, Wing-Kee Lee, Natascha A. Wolff, Barbara Storch, Szymon Żerko, Robert Konrat, Wiktor Koźmiński, Kathrin Breuker, Frank Thévenod, Nicolas Coudevylle


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The neutrophil gelatinase associated lipocalin (NGAL, aslo known as LCN2) and its cellular receptor (LCN2-R) are involved in many physiological and pathological processes such as cell differentiation, apoptosis and inflammation. These pleiotropic functions mainly rely on NGALs siderophore mediated iron transport properties. However the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo-state to form a fuzzy complex. The relatively weak affinity (≈ 10μM) between hLCN2-R-NTD and apoNGAL suggests that the N-terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, hLCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor, or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.

 

New Article in Journal of Biomolecular NMR

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High resolution 4D HPCH experiment for sequential assignment of 13C-labeled RNAs via phosphodiester backbone

Saurabh Saxena, Jan Stanek, Mirko Cevec, Janez Plavec, Wiktor Koźmiński


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The three-dimensional structure determination of RNAs by NMR spectroscopy requires sequential resonance assignment, often hampered by assignment ambiguities and limited dispersion of 1H and 13C chemical shifts, especially of C4′/H4′. Here we present a novel through-bond 4D HPCH NMR experiment involving phosphate backbone where C4′–H4′ correlations are resolved along the 1H3′–31P spectral planes. The experiment provides high peak resolution and effectively removes ambiguities encountered during assignments. Enhanced peak dispersion is provided by the inclusion of additional 31P and 1H3′ dimensions and constant-time evolution of chemical shifts. High spectral resolution is obtained by using non-uniform sampling in three indirect dimensions. The experiment fully utilizes the isotopic 13C-labeling with evolution of C4′ carbons. Band selective 13C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the C4′–C3′ and C4′–C5′ homonuclear couplings. Multiple quantum line narrowing is employed to minimize sensitivity loses. The 4D HPCH experiment is verified and successfully applied to a non-coding 34-nt RNA consisting typical structure elements and a 14-nt RNA hairpin capped by cUUCGg tetraloop.

 
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