Project: ‘New tools and applications of NMR spectroscopy beyond resolution limitation.’
Project coordinator: prof. Wiktor Koźmiński.
Project duration: 2016 - 2021.
Positions for MSc students and Postdoc available.
The aim of the project is an expansion of capabilities of the high-resolution nuclear magnetic resonance (NMR) spectroscopy which is a fundamental tool of modern structural biology. The structure and dynamics of proteins will be studied using new spectral parameters, such as cross-correlated relaxation rates. The research conducted in the frames of the project will make use of multidimensional NMR spectroscopy of isotopically enriched samples (13C, 15N, 2H) of proteins, both of folded and disordered nature. In addition, high hydrostatic pressure NMR will be employed to study conformational equilibria and dynamics of investigated proteins. Exceptionally high-resolution of 4 and 5 dimensional spectra will be achieved thanks to non-uniform sampling and advanced processing tools.
All positions are available from June, 2016. Candidate for MSc position shoud hold BSc preferably in chemistry, physics, biology or computer-science. All stipends are funded from NCN MAESTRO grant.
Postdoc candidates are asked to directly contact project coordinator.
More info: prof. Wiktor Koźmiński,
Application deadline: ongoing recruitment.
New Article in Journal of the American Chemical Society
Maria Baias, Pieter E. S. Smith, Koning Shen, Lukasz A. Joachimiak, Szymon Żerko, Wiktor Koźmiński, Judith Frydman, Lucio Frydman
Many neurodegenerative diseases are characterized by misfolding and aggregation of an expanded polyglutamine tract (polyQ). Huntington’s Disease, caused by expansion of the polyQ tract in exon 1 of the Huntingtin protein (Htt), is associated with aggregation and neuronal toxicity. Despite recent structural progress in understanding the structures of amyloid fibrils, little is known about the solution states of Htt in general, and about molecular details of their transition from soluble to aggregation-prone conformations in particular. This is an important question, given the increasing realization that toxicity may reside in soluble conformers. This study presents an approach that combines NMR with computational methods to elucidate the structural conformations of Htt Exon 1 in solution. Of particular focus was Htt’s N17 domain sited N-terminal to the polyQ tract, which is key to enhancing aggregation and modulate Htt toxicity. Such in-depth structural study of Htt presents a number of unique challenges: the long homopolymeric polyQ tract contains nearly identical residues, exon 1 displays a high degree of conformational flexibility leading to a scaling of the NMR chemical shift dispersion, and a large portion of the backbone amide groups are solvent-exposed leading to fast hydrogen exchange and causing extensive line broadening. To deal with these problems, NMR assignment was achieved on a minimal Htt exon 1, comprising the N17 domain, a polyQ tract of 17 glutamines, and a short hexameric polyProline region that does not contribute to the spectrum. A pH titration method enhanced this polypeptide’s solubility and, with the aid of ≤5D NMR, permitted the full assignment of N17 and the entire polyQ tract. Structural predictions were then derived using the experimental chemical shifts of the Htt peptide at low and neutral pH, together with various different computational approaches. All these methods concurred in indicating that low-pH protonation stabilizes a soluble conformation where a helical region of N17 propagates into the polyQ region, while at neutral pH both N17 and the polyQ become largely unstructured—thereby suggesting a mechanism for how N17 regulates Htt aggregation.
Congratulations to Saurabh Saxena on defending Ph.D. thesis entitled New NMR experiments for nucleic acids and intrinsically disordered proteins.
New Article in Journal of Biomolecular NMR
Szymon Żerko, Piotr Byrski, Paweł Włodarczyk-Pruszyński, Michał Górka, Karin Ledolter, Eliezer Masliah, Robert Konrat, Wiktor Koźmiński
New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.
New Article in Journal of Biomolecular NMR
Alessandro Piai, Leonardo Gonnelli, Isabella C. Felli, Roberta Pierattelli, Krzysztof Kazimierczuk, Katarzyna Grudziąż, Wiktor Koźmiński, Anna Zawadzka-Kazimierczuk
Resonance assignment is a prerequisite for almost any NMR-based study of proteins. It can be very challenging in some cases, however, due to the nature of the protein under investigation. This is the case with intrinsically disordered proteins, for example, whose NMR spectra suffer from low chemical shifts dispersion and generally low resolution. For these systems, sequence specific assignment is highly time-consuming, so the prospect of using automatic strategies for their assignment is very attractive. In this article we present a new version of the automatic assignment program TSAR dedicated to intrinsically disordered proteins. In particular, we demonstrate how the automatic procedure can be improved by incorporating methods for amino acid recognition and information on chemical shifts in selected amino acids. The approach was tested in silico on 16 disordered proteins and experimentally on α-synuclein, with remarkably good results.
New Article in Scientific Reports
Tomasz Kochańczyk, Michał Nowakowski, Dominika Wojewska, Andrzej Ejchart, Wiktor Koźmiński, Artur Krężel
The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.
A to B Transition
New Article in Journal of Biological Chemistry
Ana-Isabel Cabedo Martinez, Katharina Weinhaupl, Wing-Kee Lee, Natascha A. Wolff, Barbara Storch, Szymon Żerko, Robert Konrat, Wiktor Koźmiński, Kathrin Breuker, Frank Thévenod, Nicolas Coudevylle
The neutrophil gelatinase associated lipocalin (NGAL, aslo known as LCN2) and its cellular receptor (LCN2-R) are involved in many physiological and pathological processes such as cell differentiation, apoptosis and inflammation. These pleiotropic functions mainly rely on NGALs siderophore mediated iron transport properties. However the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo-state to form a fuzzy complex. The relatively weak affinity (≈ 10μM) between hLCN2-R-NTD and apoNGAL suggests that the N-terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, hLCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor, or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.
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