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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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.

 

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


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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.

 

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.

 


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