We have developed experimental methods for probing time-dependent structural conversion processes in biomolecular systems on the millisecond time scale. In these “time-resolved solid state NMR” experiments, a process of interest is initiated by rapid mixing of two solutions or by a rapid "inverse temperature jump", both of which can be accomplished in one millisecond or less. After a variable evolution period for structural evolution, the solution is frozen in less than one millisecond to trap transient intermediate states. Low-temperature, DNP-enhanced solid state NMR is then used to extract molecular structural information.
Previous studies used time-resolved solid state NMR to characterize tetramerization process of melittin, a 26-residue peptide from bee venom, following either a rapid pH jump (Jeon et al., Proc. Natl. Acad. Sci. 2019) or a rapid temperature drop (Wilson and Tycko, J. Am. Chem. Soc. 2022), and to characterize the process by which calmodulin forms complexes with a target peptide in the presence of Ca2+ (Jeon et al., J. Am. Chem. Soc. 2020; Schmidt et al., Proc. Natl. Acad. Sci. 2022).
In one recent study, we used a combination of time-resolved solid state NMR and time-resolved light scattering to examine oligomerization by the 40-residue amyloid-β peptide (Aβ40), initiated by a rapid pH drop from 12.0 to 7.4 (Jeon et al., Nature Commun. 2023). The combined data reveal that Aβ40 molecules undergo a large change in conformational preferences, from random-coil to β-strand, in less than one millisecond, even before a significant population of dimers appears. Subsequent conformational changes are minor for many minutes, even as oligomers grow to sizes exceeding 100 molecules.
In another recent study, we applied time-resolved solid state NMR with rapid inverse temperature-jump methods to the folding process of HP35, a well-studied model system for protein folding that has been shown previously to fold within 10 ms. While the NMR data are consistent with prior studies, they also reveal that the microsecond-timescale main folding event is followed by a millisecond-timescale annealing process in which the conformations and packing of amino acid sidechains in the HP35 core is optimized (Wilson et al., manuscript under review).
Ongoing studies apply similar techniques to other classes of processes. For example, preliminary results from studies of liquid-liquid phase separation by low-complexity protein domains and studies of DNA hybridization may be presented.