Jul 1, 1999

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

Protein Science : a Publication of the Protein Society
T R KillickA R Fersht


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 beta-sheet region including residues Tyr47, Ala82, Thr85, and Val50.

Mentioned in this Paper

In Vivo NMR Spectroscopy
Bacterial Proteins
Fluorescence Spectroscopy
Bacillus amyloliquefaciens ribonuclease
Protein Conformation
Urea Measurement

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