Millisecond Time-Resolved Solid-State NMR Initiated by Rapid Inverse Temperature Jumps

Elucidation of the detailed mechanisms by which biological macromolecules undergo major structural conversions, such as folding, complex formation, and self-assembly, is a central concern of biophysical chemistry that will benefit from new experimental methods. We describe a simple technique for initiating a structural conversion process by a rapid decrease in the temperature of a solution, i.e., a rapid inverse temperature jump. By pumping solutions through copper capillary tubes that are thermally anchored to heated and cooled blocks, solution temperatures can be switched from 95 to 30 °C (or lower) in about 0.8 ms. For time-resolved solid-state nuclear magnetic resonance (ssNMR), solutions can then be frozen rapidly by spraying into cold isopentane after a variable structural evolution time τ_e. As an initial demonstration, we use this "inverse T-jump" technique to characterize the kinetics and mechanism by which the 26-residue peptide melittin converts from its primarily disordered, monomeric state at 95 °C to its α-helical, tetrameric state at 30 °C. One- and two-dimensional ssNMR spectra of frozen solutions with various values of τ_e, recorded at 25 K with signal enhancements from dynamic nuclear polarization, show that both helical secondary structure and intermolecular contacts develop on the same time scale of about 6 ms. The dependences on τ_e of both intraresidue crosspeak patterns and inter-residue crosspeak volumes in two-dimensional spectra can be fit with a unidirectional dimerization model, consistent with dimerization being the rate-limiting step for melittin tetramer formation.