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Real‐time nmr studies on a transient folding intermediate of barstar
Author(s) -
Killick Thomas R.,
Freund STEFAN M. V.,
Fersht Alan R.
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.6.1286
Subject(s) - chemistry , barnase , heteronuclear single quantum coherence spectroscopy , folding (dsp implementation) , nuclear magnetic resonance spectroscopy , stereochemistry , phi value analysis , peptide bond , crystallography , protein folding , isomerase , alanine , kinetics , enzyme , amino acid , biochemistry , ribonuclease , rna , physics , quantum mechanics , electrical engineering , gene , engineering
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 1 H, 15 N 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.