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.