Vitali Tugarinov

NMR methodology is developed for accurate measurements of solvent exchange rates in asparagine and glutamine side-chain NH₂ groups of proteins by z-exchange spectroscopy. A rigorous model of solvent exchange in NH₂ groups of proteins dissolved in a 50%/50% (v/v) mixture of H₂O/D₂O solvents, that incorporates the independently measured rates of hindered rotation around the carboxamide C′–N bond, allowed determining solvent exchange rates for side-chain amides of the model proteins ubiquitin and GB1, as well as a subset of NH₂ groups of a buried cavity mutant of T4 lysozyme. In agreement with earlier findings, the amide protons at E (anti) positions usually exchange faster with the solvent than their Z (syn) counterparts. The temperature dependence of the rates of hindered rotation and solvent exchange in ubiquitin showed that the two processes are enthalpically driven and characterized by very similar activation enthalpies of ∼70 kJ/mol.

Complete degeneracy of aliphatic ¹³ C chemical shifts of the same type of amino‐acid residues precludes unambiguous NMR assignments of asparagine (Asn) and glutamine (Gln) carboxamide NH ₂ groups in intrinsically disordered proteins with conventional techniques. Here, a pair of triple‐resonance NMR experiments is described that correlate the ¹ H/ ¹⁵ N chemical shifts of Asn and Gln carboxamide NH ₂ groups with the backbone amide ¹⁵ N chemical shifts of the same and the next residue. These experiments exploit a by far superior dispersion of backbone amide ¹⁵ N chemical shifts, and permit unambiguous assignments of all Asn/Gln side‐chain NH ₂ groups in the intrinsically disordered protein α ‐synuclein and the majority of Gln NH ₂ groups in the disordered huntington exon‐1 protein with a 7‐residue glutamine repeat, htt ^ex1 Q ₇ .

We show that water saturation leads to deleterious losses in sensitivity of methyl signals in selectively methyl-[ ¹³ CH ₃ ]-labeled protein samples of high molecular weight proteins dissolved in H ₂ O. These losses arise from efficient cross-relaxation between methyl protons and proximal labile protons in the protein structure. A phenomenological model for analysis of methyl intensity decay profiles that involves exchange saturation transfer of magnetization from localized proton spins of water to various labile groups in the protein structure that, in turn, efficiently cross-relax with protons of methyl groups, is described. Analysis of methyl intensity decay profiles with this model allows cross-relaxation rates (σ) between methyl and labile protons to be determined and permits identification of methyl sites in close proximity to labile groups in the protein structure.

Closed-form expressions for the analysis of Dark state Exchange Saturation Transfer (DEST) NMR experiments, a powerful experimental tool for characterizing exchange processes involving the interaction of NMR visible species with very high molecular weight partners, is presented. Essentially identical exchange and relaxation parameters are derived from the analytical and numerical best fits of the DEST profiles obtained for a protein construct derived from huntingtin exon-1, comprising the N-terminal amphiphilic sequence followed by a seven-residue glutamine repeat, htt^NTQ₇, in the presence of small (SUV) and large (LUV) unilamellar lipid vesicles. The use of analytical expressions significantly speeds up the fitting of experimental DEST profiles to a two-state exchange model and simplifies the analysis of the DEST effects.

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH₂ groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing ¹⁵N and ¹H components of NH₂ multiplets. Although even modest sensitivity gains in 2D NH₂-TROSY correlation maps compared to their decoupled NH₂–HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH₂ sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH₂-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH₂ positions of the L99A T4 lysozyme mutant — a model system for studies of the role of protein dynamics in ligand binding.

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hht^ex1) with a 35 polyglutamine repeat, htt^ex1Q₃₅, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of htt^ex1Q₃₅ aggregation, monitored by measuring cross-peak intensities in a series of 2D ¹H-¹⁵N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" htt^ex1Q₃₅ tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, htt^ex1Q₇, lend confidence to the conclusions drawn from the fits to the htt^ex1Q₃₅ aggregation kinetics.

Pathogenic huntingtin exon‐1 protein (htt^ex1), characterized by an expanded polyglutamine tract located between the N‐terminal amphiphilic region and a C‐terminal polyproline‐rich domain, forms fibrils that accumulate in neuronal inclusion bodies, and is associated with a fatal, autosomal dominant neurodegenerative condition known as Huntington's disease. Here a complete kinetic model is described for aggregation/fibril formation of a htt^ex1 construct with a 35‐residue polyglutamine repeat, htt^ex1Q₃₅. Using exchange NMR spectroscopy, it is previously shown that the reversible formation of a sparsely‐populated tetramer of the N‐terminal amphiphilic domain of htt^ex1Q₃₅, comprising a D₂ symmetric four‐helix bundle, occurs on the microsecond time‐scale and is a prerequisite for subsequent nucleation and fibril formation on a time scale that is many orders of magnitude slower (hours). Here a unified kinetic model of htt^ex1Q₃₅ aggregation is developed in which fast, reversible tetramerization is directly linked to slow irreversible fibril formation via conversion of pre‐equilibrated tetrameric species to “active”, chain elongation‐capable nuclei by conformational re‐arrangement with a finite, monomer‐independent rate. The unified model permits global quantitative analysis of reversible tetramerization and irreversible fibril formation from a time series of ¹H‐¹⁵N correlation spectra recorded during the course of htt^ex1Q₃₅ aggregation.