Application of Equilibrium and Stopped-Flow ¹⁹F NMR Spectroscopy to Protein Folding: Studies of E. coli Dihydrofolate Reductase

This chapter describes the application of equilibrium and stopped-flow ¹⁹F nuclear magnetic resonance (NMR) spectroscopy to protein folding. It presents ¹⁹F NMR spectra of dihydrofolate reductase at five time intervals after unfolding was initiated with urea. In an experiment described in the chapter, the initial delay after injection was 1.5 s to allow the lock to stabilize and to orient the spins in the magnetic field. One fid was collected at 60 time points following each injection, and the experiment was repeated 18 times. The smooth lines at each time interval represent a model calculated by Bayesian analysis of the time domain data. The resonances of both native and unfolded protein could be seen in the first transients. A substantial amount of native peak intensity disappeared prior to the first transient at 1.5 s, while a large increase in denatured peak intensity occurred after 14 s. The use of ¹⁹F NMR to examine the equilibrium and kinetic folding behavior of fluorine labeled proteins may provide structural information about protein folding by examining side-chain behavior during the folding process.