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 been restarted. Challenges in generating sufficient amounts of isotopically enriched material have motivated us to additionally study the folding pathway of the bee venom peptide melittin, which switches from a folded tetrameric structure to an unfolded state when increasing hydrostatic pressure. Comparison of these data to those of Abeta, that also appears to adopt a tetrameric state prior to forming larger oligomers is used to evaluate how uniquely the presence of such an intermediate can be established. In a separate study of protein folding, we are investigating the pathways by which a ubiquitin mutant (L50A) switches from its unfolded state to the folded state, which is virtually indistinguishable from that of the wild-type protein. Multiple meta-stable intermediates in the folding pathway of this protein can be identified on the basis of the NMR spectra. Whereas for one of the highly populated (up to 35%) intermediates, the structure can be identified as that of a "slipped" out-of-register C-terminal beta strand, a second intermediate (populated at up to ca 15%) is also present. Whereas this second intermediate is also on-pathway, its structure has not yet been identified. The L50A ubiquitin mutant represents the first case of a protein for which multiple, on pathway, meta-stable folding intermediates have been identified.