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Dysregulation of post-translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near-physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3β) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC 50 : 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC 50 : 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of 1 H, 13 C, and 15 N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.

Side chains possess a broader conformational space (compared to the backbone) and are directly affected by intra- and intermolecular interactions, hence their dynamics and the corresponding NMR relaxation data are more sensitive and informative. Nevertheless, transverse relaxation in [Formula: see text] ([Formula: see text] or [Formula: see text]) spin systems is predominantly non-measurable in uniformly [Formula: see text]-labeled proteins due to cross-correlation effects. In the present publication, we propose a number of pulse sequences for accurate and precise measurement of the dipole-dipole transverse cross-correlated relaxation rate [Formula: see text], which, similarly to [Formula: see text] measurements, provides information about the amplitudes of intramolecular dynamics. The suggested approach has allowed us to circumvent a number of obstacles that were limiting earlier applications of [Formula: see text]: (1) impossibility of transmission of the central component of the triplet of [Formula: see text] group to [Formula: see text]-acquisition via INEPT has been solved by transmission of the averaged signal of "inner" and "outer" components of the triplet; (2) direct recording of the entire triplets resulting in substantial overlap of side chain signals has been replaced by recording of individual singlets with the use of [Formula: see text]-modulated approach and constant-time evolution; (3) low sensitivity has been enhanced via proton acquisition which required special attention to a zero-quantum coherence evolution. The proposed method expands the set of "dynamics sensors" covering protein side chains and substantially improves the quality and the level of detail of experimental data describing dynamic processes in proteins and protein complexes.

In the case of soluble proteins, chemical shift mapping is used to identify the intermolecular interfaces when the NOE-based calculations of spatial structure of the molecular assembly are impossible or impracticable. However, the reliability of the membrane protein interface mapping based on chemical shifts or other relevant parameters was never assessed. In the present work, we investigate the predictive power of various NMR parameters that can be used for mapping of helix-helix interfaces in dimeric TM domains. These parameters are studied on a dataset containing three structures of helical dimers obtained for two different proteins in various membrane mimetics. We conclude that the amide chemical shifts have very little predictive value, while the methyl chemical shifts could be used to predict interfaces, though with great care. We suggest an approach based on conversion of the carbon NMR relaxation parameters of methyl groups into parameters of motion, and one of such values, the characteristic time of methyl rotation, appears to be a reliable sensor of interhelix contacts in transmembrane domains. The carbon NMR relaxation parameters of methyl groups can be measured accurately and with high sensitivity and resolution, making the proposed parameter a useful tool for investigation of protein-protein interfaces even in large membrane proteins. An approach to build the models of transmembrane dimers based on perturbations of methyl parameters and TMDOCK software is suggested.

Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain., (© 2013 Elsevier B.V. All rights reserved.)