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 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.

 

A to B Transition

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'A' state

tranA

Transition state

tranAB

'B' state

tranB

 

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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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


amino selective

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.

 


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