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 Biochemistry


Hyperphosphorylation of Human Osteopontin and Its Impact on Structural Dynamics and Molecular Recognition

Borja Mateos, Julian Holzinger, Clara Conrad-Billroth, Gerald Platzer, Szymon Żerko, Marco Sealey-Cardona, Dorothea Anrather, Wiktor Koźmiński, Robert Konrat

bi1c00050 0007

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells. Intrinsically disordered proteins (IDPs) are particular targets of PTM protein kinases due to their involvement in fundamental protein interaction networks. Despite their dynamic nature, IDPs are far from having random-coil conformations but exhibit significant structural heterogeneity. Changes in the molecular environment, most prominently in the form of PTM via phosphorylation, can modulate these structural features. Therefore, how phosphorylation events can alter conformational ensembles of IDPs and their interactions with binding partners is of great interest. Here we study the effects of hyperphosphorylation on the IDP osteopontin (OPN), an extracellular target of the Fam20C kinase. We report a full characterization of the phosphorylation sites of OPN using a combined nuclear magnetic resonance/mass spectrometry approach and provide evidence for an increase in the local flexibility of highly phosphorylated regions and the ensuing overall structural elongation. Our study emphasizes the simultaneous importance of electrostatic and hydrophobic interactions in the formation of compact substates in IDPs and their relevance for molecular recognition events.


New Article in Angewandte Chemie


Non‐Stationary Complementary Non‐Uniform Sampling (NOSCO NUS) for Fast Acquisition of Serial 2D NMR Titration Data

Javier A. Romero,  Ewa K. Nawrocka, Alexandra Shchukina, Francisco J. Blanco, Tammo Diercks, Krzysztof Kazimierczuk

anie202009479 toc 0001 m

NMR spectroscopy offers unique benefits for ligand binding studies on isotopically labelled target proteins. These benefits include atomic resolution, direct distinction of binding sites and modes, a lowest detectable affinity limit, and function independent setup. Yet, retracing protein signal assignments from apo to holo states to derive exact dissociation constants and chemical shift perturbation amplitudes (for ligand docking and structure‐based optimization) requires lengthy titration series of 2D heteronuclear correlation spectra at variable ligand concentration that may exceed the protein's lifetime and available spectrometer time. We present a novel method to overcome this critical limitation, based on non‐stationary complementary non‐uniform sampling (NOSCO NUS) combined with a robust particle swarm optimization algorithm. We illustrate its potential in two challenging studies with very distinct protein sizes and binding affinities, showing that NOSCO NUS can reduce measurement times by an order of magnitude to make such highly informative NMR titration studies more broadly feasible.


New Article in Journal of Biomolecular NMR


Dynamic 15N{1H} NOE measurements: a tool for studying protein dynamics

Vladlena Kharchenko, Michał Nowakowski, Mariusz Jaremko, Andrzej Ejchart, Łukasz Jaremko

michal dynamicNOE

Intramolecular motions in proteins are one of the important factors that determine their biological activity and interactions with molecules of biological importance. Magnetic relaxation of 15N amide nuclei allows one to monitor motions of protein backbone over a wide range of time scales. 15N{1H} nuclear Overhauser effect is essential for the identification of fast backbone motions in proteins. Therefore, exact measurements of NOE values and their accuracies are critical for determining the picosecond time scale of protein backbone. Measurement of dynamic NOE allows for the determination of NOE values and their probable errors defined by any sound criterion of nonlinear regression methods. The dynamic NOE measurements can be readily applied for non-deuterated or deuterated proteins in both HSQC and TROSY-type experiments. Comparison of the dynamic NOE method with commonly implied steady-state NOE is presented in measurements performed at three magnetic field strengths. It is also shown that improperly set NOE measurement cannot be restored with correction factors reported in the literature.


New Article in Journal of the American Chemical Society


Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition

Jan Stanek, Tobias Schubeis, Piotr Paluch, Peter Günter, Loren B. Andreas, Guido Pintacuda

ja0c00251 0006

Thanks to magic-angle spinning (MAS) probes with frequencies of 60–100 kHz, the benefit of high-sensitivity 1H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ∼20 kDa. Here, we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation, and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent data set can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully automated peak picking and resonance assignment. The method, dubbed RAVASSA (redundant assignment via a single simultaneous acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and 1 week of spectrometer time.


New Article in Angewandte Chemie


Protein NMR resonance assignment without spectral analysis: 5D SOlid‐state Automated Projection SpectroscopY (SO‐APSY)

Henry W. Orton, Jan Stanek, Tobias Schubeis, Dylan Foucaudeau, Claire Ollier, Adrian W. Draney, Tanguy Le Marchand, Diane Cala-De Paepe, Isabella C. Felli, Roberta Pierattelli, Sebastian Hiller, Wolfgang Bermel, Guido Pintacuda


Narrow proton signals, high sensitivity, and efficient coherence transfersprovided by fast magic-angle spinning at high magnetic fields make automated projection spectroscopy feasible in protein solid-state NMR. We present the first ultra-high dimensional implementation of this approach where 5D peak lists are reconstructed from a number of 2D projections for protein samples of different molecular size and aggregation state, featuring limited dispersion of chemical shifts or inhomogeneous broadenings. The resulting datasets are particularly suitable to automated analysis, yielding rapid and unbiased backbone resonance assignments.


New Article in Journal of Biomolecular NMR


A novel high-dimensional NMR experiment for resolving protein backbone dihedral angle ambiguities

Clemens Kauffmann, Krzysztof Kazimierczuk, Thomas C. Schwarz, Robert Konrat, Anna Zawadzka-Kazimierczuk

CCR Ania

Intrinsically disordered proteins (IDPs) are challenging established structural biology perception and urge a reassessment of the conventional understanding of the subtle interplay between protein structure and dynamics. Due to their importance in eukaryotic life and central role in protein interaction networks, IDP research is a fascinating and highly relevant research area in which NMR spectroscopy is destined to be a key player. The flexible nature of IDPs, as a result of the sampling of a vast conformational space, however, poses a tremendous scientific challenge, both technically and theoretically. Pronounced signal averaging results in narrow signal dispersion and requires higher dimensionality NMR techniques. Moreover, a fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution, where often the interpretation relies on the concept of ‘residual structure’ or ‘conformational preference’. An important source of structural information is information-rich NMR experiments that probe protein backbone dihedral angles in a unique manner. Cross-correlated relaxation experiments have proven to fulfil this task as they provide unique information about protein backbones, particularly in IDPs. Here we present a novel cross-correlation experiment that utilizes non-uniform sampling detection schemes to resolve protein backbone dihedral ambiguities in IDPs. The sensitivity of this novel technique is illustrated with an application to the prototypical IDP α-Synculein for which unexpected deviations from random-coil-like behaviour could be observed.


New Article in Chemistry — A European Journal


Metal exchange in interprotein Zn(II)‐binding site of Rad50 hook domain – structural insights into Cd(II)‐induced DNA repair inhibition

Michał Padjasek, Maciej Maciejczyk, Michał Nowakowski, Olga Kerber, Maciej Pyrka, Wiktor Koźmiński, Artur Krężel


Cd(II) is a major genotoxic agent that readily displaces Zn(II) in a multitude of zinc proteins, abrogates redox homeostasis and deregulates cellular metalloproteome. To date this displacement has been described mostly for cysteine‐rich intraprotein binding sites in certain zinc finger domains and metallothionein. To visualize how Zn(II) to Cd(II) swap can affect the target protein’s status and thus understand the molecular basis of Cd(II)‐induced genotoxicity we focused on an intermolecular Zn(II)‐binding site from the crucial DNA repair protein Rad50 and its zinc hook domain. Using a length‐varied peptide base we hereby demonstrate that Zn(II) to Cd(II) displacement in Rad50’s hook domain alters it in a bimodal fashion: (i) Cd(II) induces around a two‐orders‐of‐magnitude stabilization effect (log K 12 Zn(II) = 20.8 vs log K 12 Cd(II) = 22.7), which defines an extremely high affinity of a peptide towards a metal ion, and (ii) disrupts the overall assembly of the domain, as shown by NMR and anisotropy decay data. Based on our results we propose a novel model explaining the molecular mechanism of Cd(II) genotoxicity that underlines Cd(II)’s impact on Rad50’s dimer stability and quaternary structure that could potentially result in abrogation of the major DNA damage response pathway.


New Review in Journal of Inorganic Biochemistry


Towards the functional high-resolution coordination chemistry of blood plasma human serum albumin

Samah Al-Harthi, Joanna Lachowicz, Michał Nowakowski, Mariusz Jaremko, Łukasz Jaremko

1 s2.0 S0162013419301734 ga1 lrg

Human serum albumin (HSA) is a monomeric, globular, multi-carrier and the most abundant protein in the blood. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules. For decades, HSA's ability to bind to various ligands has led many scientists to study its physiological properties and protein structure; indeed, a better understanding of HSA-ligand interactions in human blood, at the atomic level, will likely foster the development of more potent, and overall more performant, diagnostic and therapeutic tools against serious human disorders such as diabetes, cardiovascular disorders, and cancer. Here, we present a concise overview of the current knowledge of HSA's structural characteristics, and its coordination chemistry with transition metal ions, within the scope and limitations of current techniques and biophysical methods to reach atomic resolution in solution and in blood serum. We also highlight the overwhelming need of a detailed atomistic understanding of HSA dynamic structures and interactions that are transient, weak, multi-site and multi-step, and allosterically affected by each other. Considering the fact that HSA is a current clinical tool for drug delivery systems and a potential contender as molecular cargo and nano-vehicle used in biophysical, clinical and industrial fields, we underline the emerging need for novel approaches to target the dynamic functional coordination chemistry of the human blood serum albumin in solution, at the atomic level.

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