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.

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 Molecules

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The Two Isoforms of Lyn Display Different Intramolecular Fuzzy Complexes with the SH3 Domain

João M. C. Teixeira, Héctor Fuentes, Stasė Bielskutė, Margarida Gairi, Szymon Żerko, Wiktor Koźmiński, Miquel Pons


The function of the intrinsically disordered Unique domain of the Src family of tyrosine kinases (SFK), where the largest differences between family members are concentrated, remains poorly understood. Recent studies in c-Src have demonstrated that the Unique region forms transient interactions, described as an intramolecular fuzzy complex, with the SH3 domain and suggested that similar complexes could be formed by other SFKs. Src and Lyn are members of a distinct subfamily of SFKs. Lyn is a key player in the immunologic response and exists in two isoforms originating from alternative splicing in the Unique domain. We have used NMR to compare the intramolecular interactions in the two isoforms and found that the alternatively spliced segment interacts specifically with the so-called RT-loop in the SH3 domain and that this interaction is abolished when a polyproline ligand binds to the SH3 domain. These results support the generality of the fuzzy complex formation in distinct subfamilies of SFKs and its physiological role, as the naturally occurring alternative splicing modulates the interactions in this complex.

 

New Article in The Journal of Physical Chemistry A

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Conformational Equilibrium of Cinchonidine in C6D12 Solution. Alternative NMR/DFT Approach

Sergey Molchanov, Tomasz Rowicki, Adam Gryff-Keller,  Wiktor Koźmiński


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1H NMR and 13C NMR chemical shifts as well as conformation dependent vicinal 1H–1H spin–spin coupling constants for cinchonidine in a dilute C6D12 solution have been measured. These data have been interpreted in detail exploiting the results of the extensive quantum chemistry calculations of molecular geometry and NMR parameters of the molecule, performed using the density functional theory (DFT) B3LYP/6-311++G(2d,p) polarizable continuum model (PCM) level of theory. The experimental values of NMR parameters for cinchonidine have been reproduced very well in terms of parameters calculated for key conformers of this molecule. Simultaneously, the analysis has provided us with a lot of information on conformational equilibrium of cinchonidine in the investigated solution. These findings remain in general agreement with the conclusions of other works, based on NOESY spectra or other physicochemical data. Thus, a careful quantitative interpretation of easily measurable NMR chemical shifts can be an independent and valuable source of structural information even in such complex cases as cinchonidine in solution.

 

New Article in Journal of Biological Inorganic Chemistry

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Structure and dynamics of Helicobacter pylori nickel-chaperone HypA: an integrated approach using NMR spectroscopy, functional assays and computational tools

Chris A. E. M. Spronk, Szymon Żerko, Michał Górka, Wiktor Koźmiński, Benjamin Bardiaux, Barbara Zambelli, Francesco Musiani, Mario Piccioli, Priyanka Basak, Faith C. Blum, Ryan C. Johnson, Heidi Hu, D. Scott Merrell, Michael Maroney, Stefano Ciuri


HypA

Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3–7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.

 

New Article in Journal of Biomolecular NMR

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Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters

Jerzy Sitkowski, Wojciech Bocian, Elżbieta Bednarek, Mateusz Urbańczyk, Wiktor Koźmiński, Piotr Borowicz, Grażyna Płucienniczak, Natalia Łukasiewicz, Iwona Sokołowska, Lech Kozerski


The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.

 
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