A protein folding intermediate of ribonuclease T-1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy

The rate-limiting step during the refolding of S54G/P55N ribonuclease T-1 i s determined by the slow trans --> cis prolyl isomerisation of Pro39. We in vestigated the refolding of this variant by one-dimensional (1D) and two-di mensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10 degrees C. Two intermediates could b e resolved with the 1D approach. The minor intermediate, which is only pres ent early during refolding, is largely unfolded. The major intermediate, wi th an incorrect trans Pro39 peptide bond, is highly structured with 33 amid e protons showing native chemical shifts and native NOE patterns. They coul d be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spect roscopy) by using a new assignment strategy to generate positive and negati ve signal intensities for native and non-native NOE cross-peaks, respective ly. Surprisingly, amide protons with nonnative environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, i ncluding the C-terminal part of the alpha-helix, beta-strands 3 and 4 and s everal loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal beta-strands 1 and 2 and the C ter minus (connected by the disulfide bonds), the N-terminal part of the alpha- helix, and the loops between beta-strands 4/5 and 5/6. Implications of thes e native and non-native structure elements of the intermediate for the refo lding of S54G/P55N ribonuclease T-1 and for cis/trans isomerizations are di scussed. (C) 1999 Academic Press.