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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“CON-CON” assignment strategy for highly flexible intrinsically disordered proteins

Alessandro Piai, Tomáš Hošek, Leonardo Gonnelli, Anna Zawadzka-Kazimierczuk, Wiktor Koźmiński, Bernhard Brutscher, Wolfgang Bermel, Roberta Pierattelli, Isabella C. Felli


CON CON

Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel 1H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on 13C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.

 

New Article in Journal of Biomolecular NMR

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C4′/H4′ selective, non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of 13C-labeled RNAs

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


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A through bond, C4′/H4′ selective, “out and stay” type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4′(i)–C4′(i)–C4′(i−1)–H4′(i−1) correlations. The 31P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4′ dimension. The experiment fully utilizes 13C-labeling of RNA by inclusion of two C4′ evolution periods. An additional evolution of H4′ is included to further enhance peak resolution. Band selective 13C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4′–C3′ and C4′–C5′ homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully 13C, 15N-labeled 34-nt hairpin RNA comprising typical structure elements.

 

Best poster at SMASH

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Mateusz Urbańczyk was awarded with best poster award at SMASH NMR!

 

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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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 Angewandte Chemie International Edition

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Accelerating Diffusion-Ordered NMR Spectroscopy by Joint Sparse Sampling of Diffusion and Time Dimensions

Mateusz Urbańczyk, Wiktor Koźmiński, Krzysztof Kazimierczuk


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Diffusion-ordered multidimensional NMR spectroscopy is a valuable technique for the analysis of complex chemical mixtures. However, this method is very time-consuming because of the costly sampling of a multidimensional signal. Various sparse sampling techniques have been proposed to accelerate such measurements, but they have always been limited to frequency dimensions of NMR spectra. It is now revealed how sparse sampling can be extended to diffusion dimensions.

 

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.
 

 


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