Real-time NMR studies on a transient folding intermediate of barstar

The refolding of barstar, the intracellular inhibitor of barnase, is domina ted by the slow formation of a cis peptidyl prolyl bond in the native prote in. The triple mutant C40/82A P27A in which two cysteine residues and one t rans proline were replaced by alanine was used as model system to investiga te the kinetics and structural consequences of the trans/cis interconversio n of Pro48. One- and two-dimensional real-time NMR spectroscopy was used to follow the trans/cis interconversion after folding was initiated by rapid dilution of the urea denatured protein. Series of H-1, N-15 HSQC spectra ac quired with and without the addition of peptidyl prolyl isomerase unambiguo usly revealed the accumulation of a transient trans-Pro48 intermediate with in the dead time of the experiment. Subtle chemical shift differences betwe en the native state and the intermediate spectra indicate that the intermed iate is predominantly native-like with a local rearrangement in the Pro48 l oop and in the beta-sheet region including residues Tyr47, Ala82, Thr85, an d Val50.