Wiktor Koźmiński's NMR group

Biological and Chemical Research Centre, University of Warsaw

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Wiktor Koźmiński's NMR Group

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


JBC nico

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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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.

 

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


SS4D jBio

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.

 

The FEBS Journal Poster Prize

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Saurabh Saxena was awarded with The FEBS Journal Poster Prize at ISMAR 2015 conference in Shanghai. Congratulations!

 

New Article in Journal of Biomolecular NMR

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13C-detected NMR experiments for automatic resonance assignment of IDPs and multiple-fixing SMFT processing

Paweł Dziekański, Katarzyna Grudziąż, Patrik Jarvoll, Wiktor Koźmiński, Anna Zawadzka-Kazimierczuk


multfixing

Intrinsically disordered proteins (IDPs) have recently attracted much interest, due to their role in many biological processes, including signaling and regulation mechanisms. High-dimensional 13C direct-detected NMR experiments have proven exceptionally useful in case of IDPs, providing spectra with superior peak dispersion. Here, two such novel experiments recorded with non-uniform sampling are introduced, these are 5D HabCabCO(CA)NCO and 5D HNCO(CA)NCO. Together with the 4D (HACA)CON(CA)NCO, an extension of the previously published 3D experiments (Pantoja-Uceda and Santoro in J Biomol NMR 59:43–50, 2014), they form a set allowing for complete and reliable resonance assignment of difficult IDPs. The processing is performed with sparse multidimensional Fourier transform based on the concept of restricting (fixing) some of spectral dimensions to a priori known resonance frequencies. In our study, a multiple-fixing method was developed, that allows easy access to spectral data. The experiments were tested on a resolution-demanding alpha-synuclein sample. Due to superior peak dispersion in high-dimensional spectrum and availability of the sequential connectivities between four consecutive residues, the overwhelming majority of resonances could be assigned automatically using the TSAR program.

 

New Article in Journal of Biomolecular NMR

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Six- and seven-dimensional experiments by combination of sparse random sampling and projection spectroscopy dedicated for backbone resonance assignment of intrinsically disordered proteins

Szymon Żerko, Wiktor Koźmiński


coh trans pathway

Two novel six- and seven-dimensional NMR experiments are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in four indirectly detected dimensions and synchronous sampling in the additional dimensions using projection spectroscopy principle. The resulted data sets could be processed as five-dimensional data using existing software. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel experiments were successfully tested using 1 mM sample of α-synuclein on 600 and 800 MHz NMR spectrometers equipped with standard room temperature probes. The experiments allowed backbone assignment from a 1-day acquisition.

 

New Review in Progress in Nuclear Magnetic Resonance Spectroscopy

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Applications of high dimensionality experiments to biomolecular NMR

Michał Nowakowski, Saurabh Saxena, Jan Stanek, Szymon Żerko, Wiktor Koźmiński


Full-size image (11 K)

High dimensionality NMR experiments facilitate resonance assignment and precise determination of spectral parameters such as coupling constants. Sparse non-uniform sampling enables acquisition of experiments of high dimensionality with high resolution in acceptable time. In this review we present and compare some significant applications of NMR experiments of dimensionality higher than three in the field of biomolecular studies in solution.

 

New Article in Biochemical Journal

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The solution structure of the MANEC-type domain from Hepatocyte Growth Factor Activator Inhibitor 1 reveals an unexpected PAN/apple domain-type fold.

Zebin Hong, Michał Nowakowski, Chris Spronk, Steen V. Petersen, Peter A Andreasen, Wiktor Koźmiński, Frans A. A. Mulder and Jan K. Jensen


manec

A decade ago, a Motif at N-terminus with Eight-Cysteines or in short MANEC was defined as a new protein domain family. This domain is found exclusively in the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor activator, all serine proteases with important roles in epithelial development, cell growth and homeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. Here we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins.

 


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