$995,227
National Institute of Diabetes and Digestive and Kidney Diseases
Maryland
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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. Using a pressure-sensitized mutant of ubiquitin (VA2-ubiquitin), we have demonstrated that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record two- and three-dimensional NMR spectra of the unfolded protein at atmospheric pressure, providing new, residue specific information on the folding process. 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. We carried out the structural characterization of a pressure-sensitized ubiquitin mutant during folding and identified an on-pathway folding intermediate with non-native beta sheet registry, previously observed to be necessary in the PINK1 mitophagy pathway. Specifically, we used fast pressure jumps, synchronized with advanced NMR measurements on the evolving ensemble of protein conformations during folding, including NOE and residual dipolar coupling interactions. We found that that non-native beta sheet hydrogen bond registry can act as a metastable trap during protein folding. This work provides a template for future investigation of metastable conformations and protein folding with rich structural detail. 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. Oligomeric states of Abeta40 were shown to exhibit a high degree of order, reflected by slow longitudinal 15N relaxation (T1 >5 s) for residues 18-21 and 31-34, whereas the N-terminal 10 residues relax much faster (T1 1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca 1000 s-1 after the oligomerization is initiated, indicating that the oligomers then have accumulated a size on the order of 1 MDa, or ca 250 peptides. Pressure-jump experiments reveal detailed information on the kinetics of the initiation of oligomer formation, as well as on the details of the structural arrangements of the peptides in the oligomer. Such experiments indicate a 3d order power dependence of the oligomerization rate on monomer concentration, indicative that a tetrameric state is the critical species forming the barrier to formation of the much larger oligomers. Experiments were interrupted by the COVID-19 pandemic but are to be resumed shortly.