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 Structural Biology

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NMR structural studies of the first catalytic half-domain of ubiquitin activating enzyme

Mariusz Jaremko, Łukasz Jaremko, Michał Nowakowski, Marek Wojciechowski, Roman H. Szczepanowski, Renata Panecka, Igor Zhukov, Matthias Bochtlerd, Andrzej Ejchart


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We report a high resolution NMR structure and 15N relaxation studies of the first catalytic cysteine half-domain (FCCH) of the mouse ubiquitin-activating enzyme E1, together with interaction studies of FCCH and the other catalytic E1 subdomain – SCCH (second catalytic cysteine half-domain). In solution, mouse FCCH forms a well-defined six-stranded antiparallel β-barrel structure, a common fold for many proteins with a variety of cellular functions. 15N relaxation data reveal FCCH complex backbone dynamics and indicate which residues experience slow intramolecular motions. Some of these residues make contacts with the polar face of ubiquitin in the co-crystal structure of yeast E1 and ubiquitin. However, the titration of FCCH with ubiquitin does not show any visible chemical shift changes in the 2D 1H/15N HSQC spectra of the FCCH. The 2D 1H/15N HSQC experiments performed both for each catalytic half-domain individually and for their equimolar mixture in the milimolar concentration range display no detectable chemical shift perturbation, suggesting a lack of interaction between the two subdomains unless they are covalently linked via the adenylation domain.

 

New Article in Journal of Biomolecular NMR

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High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins

Wolfgang Bermel, Isabella C. Felli, Leonardo Gonelli, Wiktor Koźmiński, Alessandro Piai, Roberta Pierratelli, Anna Zawadzka-Kazimierczuk


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We present three novel exclusively heteronuclear 5D 13C direct-detected NMR experiments, namely (HN-flipN)CONCACON, (HCA)CONCACON and (H)CACON(CA)CON, designed for easy sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The experiments proposed have been optimized to overcome the drawbacks which may dramatically complicate the characterization of IDPs by NMR, namely the small dispersion of chemical shifts and the fast exchange of the amide protons with the solvent. A fast and reliable automatic assignment of α-synuclein chemical shifts was obtained with the Tool for SMFT-based Assignment of Resonances (TSAR) program based on the information provided by these experiments.

 

New Review in ChemPhysChem

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High-Dimensional NMR Spectra for Structural Studies of Biomolecules

Krzysztof Kazimierczuk, Jan Stanek, Anna Zawadzka-Kazimierczuk, Wiktor Koźmiński


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Recent developments in the acquisition and processing of NMR data sets facilitate the recording of ultra-high-resolution NMR spectra in a reasonable time. The new experiments allow easy resonance assignment for folded and unfolded proteins, as well as the precise determination of spectral parameters, for example, chemical shifts, NOE contacts, coupling constants or cross-correlated relaxation rates. Owing to exceptional resolution of 4D–6D spectroscopy, detailed studies of biomolecules of unprecedented complexity are now possible. Herein, the principles of acquisition and processing methods are presented. The main applications of high-dimensional NMR experiments, including backbone and side-chain resonance assignment in proteins, as well as heteronuclear edited NOE techniques are reviewed.
 

 

New Article in Journal of Biomolecular NMR

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4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C,15N-labeled RNAs

Jan Stanek, Peter Podbevšek, Wiktor Koźmiński, Janez Plavec, Mirko Cevec


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A 4D 13C(aromatic),13C(ribose)-edited NOESY experiment is introduced to improve sequential assignment of non-coding RNA, often hampered by a limited dispersion of 1H and 13C chemical shifts. The 13C-labeling of RNA is fully utilized by inclusion of two 13C evolution periods. These dimensions provide enhanced dispersion of resonances in the 4D spectrum. High spectral resolution is obtained using random non-uniform sampling in three indirect dimensions. The autocorrelation peaks are efficiently suppressed using band-selective pulses. Since the dynamic range of observed resonances is significantly decreased, the reconstruction of the 4D spectrum is greatly simplified. The experiment can replace two conventionally sampled 3D NOESY spectra (either ribose-13C- or aromatic-13C-separated), and remove most ambiguities encountered during sequential walks. The assignment strategy based on a homonuclear and 4D C,C-edited NOESY experiments is proposed and verified on a 34-nt RNA showing typical structure elements.

 

New Article in Protein Science

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Protonation-dependent conformational variability of intrinsically disordered proteins

Leonhard Geist, Morkos A. Henen, Sandra Haiderer, Thomas C. Schwarz, Dennis Kurzbach, Anna Zawadzka-Kazimierczuk, Saurabh Saxena, Szymon Żerko, Wiktor Koźmiński, Dariush Hinderberger, Robert Konrat


Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity and undergo rearrangements of the time-averaged conformational ensemble upon changes of environmental conditions (e.g., in ionic strength, pH, molecular crowding). In contrast to stably folded proteins, IDPs often form compact conformations at acidic pH. The biological relevance of this process was, for example, demonstrated by NMR studies of the aggregation prone (low pH) state of α-Synuclein. Here we report a large-scale analysis of the pH dependence of disordered proteins using the recently developed meta-structure approach. The meta-structure analysis of a large set of intrinsically disordered proteins revealed a significant tendency of IDPs to form α-helical secondary structure elements and to preferentially fold into more compact structures under acidic conditions. The predictive validity of this novel approach was demonstrated with applications to the tumor-suppressor BASP1 and the transcription factor Tcf4.

 


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