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 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.

 

New Article in Biochemistry

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Impact of Calcium Binding and Thionylation of S100A1 Protein on Its Nuclear Magnetic Resonance-Derived Structure and Backbone Dynamics

Michał Nowakowski, Katarzyna Ruszczyńska-Bartnik, Monika Budzińska, Łukasz Jaremko, Mariusz Jaremko, Konrad Zdanowski, Andrzej Bierzyński, Andrzej Ejchart


 

S100 proteins play a crucial role in multiple important biological processes in vertebrate organisms acting predominantly as calcium signal transmitters. S100A1 is a typical representative of this family of proteins. After four Ca2+ ions bind, it undergoes a dramatic conformational change, resulting in exposure, in each of its two identical subunits, a large hydrophobic cleft that binds to target proteins. It has been shown that abnormal expression of S100A1 is strongly correlated with a number of severe human diseases: cardiomyopathy and neurodegenerative disorders. A few years ago, we found that thionylation of Cys 85, the unique cysteine in two identical S100A1 subunits, leads to a drastic increase of the affinity of the protein for calcium. We postulated that the protein activated by thionylation becomes a more efficient calcium signal transmitter. Therefore, we decided to undertake, using nuclear magnetic resonance methods, a comparative study of the structure and dynamics of native and thionylated human S100A1 in its apo and holo states. In this paper, we present the results obtained for both forms of this protein in its holo state and compare them with the previously published structure of native apo-S100. The main conclusion that we draw from these results is that the increased calcium binding affinity of S100A1 upon thionylation arises, most probably, from rearrangement of the hydrophobic core in its apo form.

 

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 Polyhedron

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Sialorphin and its analog as ligands for copper(II) ions

Elżbieta Kamysz, Aleksandra Kotynia, Żaneta Czyżnikowska, Mariusz Jaremko, Łukasz Jaremko, Michał Nowakowski, Justyna Brasun


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In this study the sialorphin (Gln-His-Asn-Pro-Arg) and its analog (Glp-His-Asn-Pro-Arg) were analyzed in terms of metal binding ability. Both peptides were synthesized using the solid-phase method. The application of number analytical methods: potentiometry, spectroscopy (UV–Vis, CD, NMR) and mass spectrometry allowed for a detailed characterization of the coordination abilities of presented peptides. The analysis of the obtained results has shown that both peptides are able to form a series of complexes. However due to the presence of free N-terminal amino group the sialorphin is more effective in metal ion binding. Nevertheless, in basic conditions both peptides involve the amide nitrogen belonging to the side chain of Asn3 moiety and form 4N complex with square planar structure. This unusual ability has been confirmed by the results obtained from the NMR studies.

 

New Article in Analytical Chemistry

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Iterative Thresholding Algorithm for Multiexponential Decay Applied to PGSE NMR Data

Mateusz Urbańczyk, Diana Bernin, Wiktor Koźmiński, Krzysztof Kazimierczuk


 

Pulsed gradient spin echo (PGSE) is a well-known NMR technique for determining diffusion coefficients. Various signal processing techniques have been introduced to solve the task, which is especially challenging when the decay is multiexponential with an unknown number of components. Here, we introduce a new method for the processing of such types of signals. Our approach modifies the Tikhonov’s regularization, known previously in CONTIN and Maximum Entropy (MaxEnt) methods, by using the -norm penalty function. The modification enforces sparsity of the result, which improves resolution, compared to both mentioned methods. We implemented the Iterative Thresholding Algorithm for Multiexponential Decay (ITAMeD), which employs the -norm minimization, using the Fast Iterative Shrinkage Thresholding Algorithm (FISTA). The proposed method is compared with the Levenberg–Marquardt-Fletcher fitting, Non-negative Least Squares (NNLS), CONTIN, and MaxEnt methods on simulated datasets, with regard to noise vulnerability and resolution. Also, the comparison with MaxEnt is presented for the experimental data of polyethylene glycol (PEG) polymer solutions and mixtures of these with various molecular weights (1080 g/mol, 11 840 g/mol, 124 700 g/mol). The results suggest that ITAMeD may be the method of choice for monodispersed samples with “discrete” distributions of diffusion coefficients.

 


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