Wiktor Koźmiński's NMR group

Biological and Chemical Research Centre, University of Warsaw

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Wiktor Koźmiński's NMR Group

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 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.

 

New Article in Angewandte Chemie International Edition

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Probing Local Backbone Geometries in Intrinsically Disordered Proteins by Cross-Correlated NMR Relaxation

Jan Stanek, Saurabh Saxena, Leonhard Geist, Robert Konrat, Wiktor Koźmiński


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An ultra-high-resolution NMR experiment for the measurement of intraresidue 1H(i)–15N(i)–13C'(i) dipolar–chemical shift anisotropy relaxation interference is employed to extract information about local backbone geometries in intrinsically disordered proteins. The study of tumor suppressor BASP1 revealed a population shift of β-turn geometries at low pH conditions and a compaction of the BASP1 structural ensemble.

 

New Article in Journal of Inclusion Phenomena and Macrocyclic Chemistry

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Complex formation of fenchone with α-cyclodextrin: NMR titrations

Michał Nowakowski, Andrzej Ejchart


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13C NMR titration studies of inclusion complexes of bicyclic terpenoid, fenchone enantiomers with α-cyclodextrin revealed their 1:2 guest–host stoichiometry. Sequential binding constants were determined indicating a strong binding cooperativity of two α-cyclodextrin to fenchone. The overall association constants were used to calculate the Gibbs free energies of diastereomeric complex formation, which might be used as a measure of chiral recognition of fenchone by α-cyclodextrin. These results were compared with corresponding data derived for camphor, which is an isomeric bicyclic terpenoid.

 

New Article in Journal of Biomolecular NMR

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Selective diagonal-free 13C,13C-edited aliphatic–aromatic NOESY experiment with non-uniform sampling

Jan Stanek, Michał Nowakowski, Saurabh Saxena, Katarzyna Ruszczyńska-Bartnik, Andrzej Ejchart, Wiktor Koźmiński


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A band-selective aromatic–aliphatic C,C-edited four-dimensional NOESY experiment is proposed here. Its key advantage is the absence of auto-correlation signals which makes it very attractive for joint use with non-uniform sampling. It is demonstrated here that the sensitivity of the experiment is not significantly affected by utilization of selective pulses (for either aromatic-13C or aliphatic-13C spins). The method was applied to the sample of E32Q mutant of human S100A1 protein, a homodimer of total molecular mass ~20 kDa. High-resolution 4D spectra were obtained from ~1.5 % of sampling points required conventionally. It is shown that superior resolution facilitates unambiguous assignment of observed aliphatic–aromatic cross-peaks. Additionally, the addition of aliphatic-13C dimension enables to resolve peaks with degenerated aliphatic 1H chemical shifts. All observed cross-peaks were validated against previously determined 3D structure of E32Q mutant of S100A1 protein (PDB 2LHL). The increased reliability of structural constraints obtained from the proposed high-resolution 4D 13C(ali),13C(aro)-edited NOESY can be exploited in the automated protocols of structure determination of proteins.

 


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