Study of protein folding and misfolding by NMR spectroscopy

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

In close collaboration with Philip Anfinrud, novel hardware was designed and developed that demonstrates, for the first time, that it is readily possible to monitor the folding of the protein chain in a residue-specific manner upon jumping the applied pressure. Pressure changes of up to 2.5 kbar, requiring 1-2 ms, are feasible and compatible with the recording of high quality NMR data. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Protein folding, as commonly portrayed, is an exploration of a rough, high-dimensional landscape ending with a final descent into a low-energy folded state. During that journey, the protein may visit shallow basins corresponding to metastable structures, potentially of biological significance. Structural characterization of metastable states has remained challenging because of their low populations, which limit traditional NMR, and short lifetimes that make crystallization for X-ray diffraction difficult without stabilizing mutations, covalent modifications, or the addition of antibodies. Brain tissue of Alzheimers disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Abeta40 and Abeta42. It remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (< 1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Experiments on the application of our pressure-jump apparatus to the structural study of the oligomers formed by the Abeta40 peptide were truncated by the shutdown of facility access, caused by the COVID-19 pandemic, but have restarted with the arrival of a new post-doctoral fellow. In related work, we have explored the use of chemical denaturation to probe the presence of transiently ordered species in the Abeta(1-42) peptide under regular, atmospheric pressure conditions. The H-1(N), N-15, C-13(alpha), and C-13' chemical shifts of A beta(1-40) and A beta(1-42) peptides and their M35-oxidized variants were monitored as a function of urea concentration and compared to analogous urea titrations of synthetic pentapeptides of homologous sequence. Fitting of the chemical shift titrations yielded a 10 +/- 1% population for a structured element at the C-terminus of A beta(1-42) that folds with a cooperativity of m = 0.06 kcal/mol.M. The fit also yielded the chemical shifts of the folded state and, using a database search based on our earlier work that correlates chemical shifts to protein structure, these shifts identified an antiparallel intramolecular beta-sheet for residues I32-A42, linked by a type I' beta-turn at G37 and G38. The structure is destabilized by oxidation of M35. Paramagnetic relaxation rates and two previously reported weak, medium-range NOE interactions are consistent with this transient beta-sheet. Introduction of the requisite A42C mutation and tagging with MTSL resulted in a small stabilization of this beta-sheet. Chemical shift analysis suggests a C-terminal beta-sheet may be present in A beta(1-40) too, but the turn type at G37 is not type I'. The approach to derive Transient Structure from chemical Denaturation by NMR (TSD-NMR), demonstrated by us for Abeta peptides, provides a sensitive tool for identifying the presence of lowly populated, transiently ordered elements in proteins that are considered to be intrinsically disordered, and represents a new, sensitive tool to extraction of structural data for such transiently structured elements.