2D NMR spectroscopy of refolding RNase Sa using polarization transfer from hyperpolarized water

Polarization transfer from hyperpolarized water through proton exchange is used to enhance the NMR signals of amide protons of the Ribonuclease Sa protein. Spectra of the refolding protein are measured within 6 s after dilution of the denaturant urea, at urea-dependent folding rates adjusted in the range of 0.3-0.8 s^-1. Peak patterns including a mixture of folded and unfolded protein at different ratios are observed. The changes in the observed signals indicate that each spectrum accesses a different point in the partial completion of the folding. A comparison to simulated 2D NMR spectra suggests a lower polarization transfer efficiency from water when the protein folds slowly, which may result from the molecular motions in the unfolded protein and the absence of long-range contacts. The ability to acquire 2D NMR spectra under different refolding conditions may open a new avenue for residue specific characterization of the folding process.