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The rate‐limiting steps for the folding of an antibody scFv fragment
Author(s) -
Jäger Marcus,
Plückthun Andreas
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)01350-1
Subject(s) - chemistry , kinetics , isomerization , folding (dsp implementation) , peptide bond , protein folding , peptide , native state , isomerase , stereochemistry , prolyl isomerase , protein disulfide isomerase , biophysics , crystallography , biochemistry , disulfide bond , catalysis , enzyme , biology , physics , quantum mechanics , pin1 , electrical engineering , engineering
The refolding kinetics of a single‐chain Fv (scFv) fragment, derived from the phosphorylcholine binding antibody McPC603, was investigated. Both prolyl‐peptide bonds which are cis in the native state affect the refolding kinetics of long‐term denatured protein. The rate‐limiting step is the trans→cis isomerization at the ProL95‐peptide bond, which is catalyzed by peptidyl‐prolyl‐ cis/trans ‐isomerase (PPIase), and is the prerequisite for correct V H /V L domain association. Refolding of short‐term denatured protein resulted in complex refolding kinetics, too. This kinetic heterogeneity could be ascribed to cis→trans re‐isomerization at the ProL95‐peptide bond to the wrong conformation in a folding intermediate. PPIase was shown to increase the fraction of slowly folding species, thereby competing with the fast folding of short‐term denatured scFv, having native proline conformations. A trapped intermediate is rapidly populated, and the return from this state becomes rate‐limiting.