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 The Journal of Physical Chemistry A


Conformational Equilibrium of Cinchonidine in C6D12 Solution. Alternative NMR/DFT Approach

Sergey Molchanov, Tomasz Rowicki, Adam Gryff-Keller,  Wiktor Koźmiński


1H NMR and 13C NMR chemical shifts as well as conformation dependent vicinal 1H–1H spin–spin coupling constants for cinchonidine in a dilute C6D12 solution have been measured. These data have been interpreted in detail exploiting the results of the extensive quantum chemistry calculations of molecular geometry and NMR parameters of the molecule, performed using the density functional theory (DFT) B3LYP/6-311++G(2d,p) polarizable continuum model (PCM) level of theory. The experimental values of NMR parameters for cinchonidine have been reproduced very well in terms of parameters calculated for key conformers of this molecule. Simultaneously, the analysis has provided us with a lot of information on conformational equilibrium of cinchonidine in the investigated solution. These findings remain in general agreement with the conclusions of other works, based on NOESY spectra or other physicochemical data. Thus, a careful quantitative interpretation of easily measurable NMR chemical shifts can be an independent and valuable source of structural information even in such complex cases as cinchonidine in solution.


New Article in Journal of Biomolecular NMR


Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters

Jerzy Sitkowski, Wojciech Bocian, Elżbieta Bednarek, Mateusz Urbańczyk, Wiktor Koźmiński, Piotr Borowicz, Grażyna Płucienniczak, Natalia Łukasiewicz, Iwona Sokołowska, Lech Kozerski

The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.


New Article in Methods


High-dimensional NMR methods for intrinsically disordered proteins studies

Katarzyna Grudziąż, Anna Zawadzka-Kazimierczuk, Wiktor Koźmiński


Intrinsically disordered proteins (IDPs) are getting more and more interest of the scientific community. Nuclear magnetic resonance (NMR) is often a technique of choice for these studies, as it provides atomic-resolution information on structure, dynamics and interactions of IDPs. Nonetheless, NMR spectra of IDPs are typically extraordinary crowded, comparing to those of structured proteins. To overcome this problem, high-dimensional NMR experiments can be used, which allow for a better peak separation. In the present review different aspects of such experiments are discussed, from data acquisition and processing to analysis, focusing on experiments for resonance assignment.


New Article in Journal of Biomolecular NMR


Fast evaluation of protein dynamics from deficient 15N relaxation data

Łukasz Jaremko, Mariusz Jaremko, Andrzej Ejchart, Michał Nowakowski


Simple and convenient method of protein dynamics evaluation from the insufficient experimental 15N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse 15N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N–H vectors on two different time scales, S2 and Rex, can be elucidated. The generalized order parameter, S2, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, Rex, identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.


New Article in Chirality


Size makes a difference: Chiral recognition in complexes of fenchone with cyclodextrins studied by means of NMR titration

Karolina Dudzik, Jacek Wójcik, Andrzej Ejchart, Michał Nowakowski

chirality MN

Gibbs energies of complex formation between enantiomers of bicyclic terpenoid, fenchone, and naturally occurring cyclodextrins, βCD and γCD, were determined by means of 13C and 1H nuclear magnetic resonance (NMR) titration data. These results were compared with the corresponding data obtained previously for the diastereomeric fenchone/αCD complexes. The size of the inner cavity of host molecules significantly influences stoichiometry, association constants, and enantiomeric differentiation of the studied complexes. These complementary data allow us to discuss qualitatively the influence of the host size on the guest–host interactions. A method of the simultaneous use of titration data collected for several resonances of different isotopes in the determination of association constants was worked out and thoroughly analyzed. Comparison of the results of global data analyses with weighted means of individual ones revealed that both these approaches are equally trustworthy.


Sympozjum sekcji NMR Polskiego Towarzystwa Chemicznego 2018


Sympozjum sekcji Rezonansu Magnetycznego Polskiego Towarzystwa Chemicznego odbędzie się w dniach 19-20 kwietnia 2018, w Centrum Nauk Biologiczno-Chemicznych Uniwersytetu Warszawskiego.


 Bruker logo







11.00 - 11.15 prof. dr hab. Wiktor Koźmiński Otwarcie
11.15 - 11.45 dr Tomasz Ratajczyk Wzmocnienie sygnału NMR biomolekuł metodą hiperpolaryzacji jądrowej indukowanej parawodorem
11.45 - 12.15 dr Igor Zhukow Badania struktury oraz dynamiki ludzkiego prionowego PrPC za pomocą spektroskopii NMR wysokiej zdolności rozdzielczej
12.15 - 12.45   Przerwa kawowa (sponsor: Bruker logo)
12.45 - 13.15 mgr Paweł Ołówek Najnowsze możliwości w dziedzinie spektrometrii NMR – JEOL
13.15 - 14.00 mgr Katarzyna Grudziąż Jak robić białka na NMR
14.00 - 15.40   Lunch (sponsor: Bruker logo)
15.30 - 15.50 mgr Maciej Kostrzewa Mechanochemiczne otrzymywanie solwatów i kokryształów Apremilastu stymulowane π-filowym rozpoznaniem molekularnym w ciele stałym
15.50 - 16.10 dr Tomasz Pawlak Badania dynamiki molekularnej za pomocą spektroskopii NMR w ciele stałym w steroidowych rotorach molekularnych
16.10 - 16.35 mgr Dariusz Gołowicz Swept Coherence Transfer - A New Approach to Quantitative 2D NMR
16.35 - ...   Relaks na Tarasie CNBCh (sponsor: LOGO)


10.00 - 10.30 prof. dr hab. Jarosław Jaźwiński Związki kompleksowe rodu(II) z pochodnymi aminokwasów - nowe receptory ligandów organicznych
10.30 - 11.00 dr Witold Andrałojć Unraveling the structural basis for the exceptional stability of RNA G-quadruplexes capped by the GGU sequence at the 3’ terminus
11.00 - 11.30   Przerwa kawowa (sponsor: Bruker logo)
11.30 - 11.45 dr Katarzyna Trzeciak Wpływ stereochemii i chemicznej modyfikacji C-końca dermorfiny na oddziaływania peptyd fosfolipid
11.45 - 12.00 dr hab. Dariusz Pisklak Zastosowanie spektroskopii NMR w fazie stałej w badaniach stałych form leków
12.00 - 12.30 dr Beata Naumczuk Regioselektywność reakcji alkilowania modelowych nukleozydów przez pochodne SN38
12.30 - 14.15 dr Piotr Garbacz Magnetyczny rezonans jądrowy w polu elektrycznym
14.15 - 14.30 prof. dr hab. Wiktor Koźmiński Zamknięcie
14.30 - ...   Lunch (sponsor: Bruker logo)





New Article in International Journal of Biological Macromolecules


Spatial attributes of the four-helix bundle group of bacteriocins – The high-resolution structure of BacSp222 in solution

Michał Nowakowski, Łukasz Jaremko, Benedykt Wladyka, Grzegorz Dubin, Andrzej Ejchart, Paweł Mak


BacSp222 is a multifunctional bacteriocin produced by Staphylococcus pseudintermedius strain 222, an opportunistic pathogen of domestic animals. At micromolar concentrations, BacSp222 kills Gram-positive bacteria and is cytotoxic toward mammalian cells, while at nanomolar doses, it acts as an immunomodulatory factor, enhancing nitric oxide release in macrophage-like cell lines. The bacteriocin is a cationic, N-terminally formylated, 50-amino-acid-long linear peptide that is rich in tryptophan residues. In this study, the solution structure of BacSp222 was determined and compared to the currently known structures of similar bacteriocins. BacSp222 was isolated from a liquid culture medium in a uniformly 13C- and 15N-labeled form, and NMR data were collected. The structure was calculated based on NMR-derived constraints and consists of a rigid and tightly packed globular bundle of four alpha-helices separated by three short turns. Although the amino acid sequence of BacSp222 has no significant similarity to any known peptide or protein, a 3D structure similarity search indicates a close relation to other four-helix bundle-motif bacteriocins, such as aureocin A53, lacticin Q and enterocins 7A/7B. Assuming similar functions, biology, structure and physicochemical properties, we propose to distinguish the four-helix bundle bacteriocins as a new Type A in subclass IId of bacteriocins, containing linear, non-pediocin-like peptides.


New Article in Frontiers in Microbiology


Fast 2D NMR for in vivo Monitoring of Bacterial Metabolism in Complex Mixtures

Rupashree Dass, Katarzyna Grudziąż, Takao Ishikawa, Michał Nowakowski, Renata Dębowska, Krzysztof Kazimierczuk

fmicb 08 01306 g001

The biological toolbox is full of techniques developed originally for analytical chemistry. Among them, spectroscopic experiments are very important source of atomic-level structural information. Nuclear magnetic resonance (NMR) spectroscopy, although very advanced in chemical and biophysical applications, has been used in microbiology only in a limited manner. So far, mostly one-dimensional 1H experiments have been reported in studies of bacterial metabolism monitored in situ. However, low spectral resolution and limited information on molecular topology limits the usability of these methods. These problems are particularly evident in the case of complex mixtures, where spectral peaks originating from many compounds overlap and make the interpretation of changes in a spectrum difficult or even impossible. Often a suite of two-dimensional (2D) NMR experiments is used to improve resolution and extract structural information from internuclear correlations. However, for dynamically changing sample, like bacterial culture, the time-consuming sampling of so-called indirect time dimensions in 2D experiments is inefficient. Here, we propose the technique known from analytical chemistry and structural biology of proteins, i.e., time-resolved non-uniform sampling. The method allows application of 2D (and multi-D) experiments in the case of quickly varying samples. The indirect dimension here is sparsely sampled resulting in significant reduction of experimental time. Compared to conventional approach based on a series of 1D measurements, this method provides extraordinary resolution and is a real-time approach to process monitoring. In this study, we demonstrate the usability of the method on a sample of Escherichia coli culture affected by ampicillin and on a sample of Propionibacterium acnes, an acne causing bacterium, mixed with a dose of face tonic, which is a complicated, multi-component mixture providing complex NMR spectrum. Through our experiments we determine the exact concentration and time at which the anti-bacterial agents affect the bacterial metabolism. We show, that it is worth to extend the NMR toolbox for microbiology by including techniques of 2D z-TOCSY, for total “fingerprinting” of a sample and 2D 13C-edited HSQC to monitor changes in concentration of metabolites in selected metabolic pathways.


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