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

Abstract

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.