The refolding of barstar, the intracellular inhibitor
of barnase, is dominated by the slow formation of a cis
peptidyl prolyl bond in the native protein. The triple
mutant C40/82A P27A in which two cysteine residues and
one trans proline were replaced by alanine was
used as model system to investigate the kinetics and structural
consequences of the trans/cis interconversion
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 1H, 15N
HSQC spectra acquired with and without the addition of
peptidyl prolyl isomerase unambiguously revealed the accumulation
of a transient trans-Pro48 intermediate within
the dead time of the experiment. Subtle chemical shift
differences between the native state and the intermediate
spectra indicate that the intermediate is predominantly
native-like with a local rearrangement in the Pro48 loop
and in the β-sheet region including residues Tyr47,
Ala82, Thr85, and Val50.