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Characterization of a quaternary‐structured folding intermediate of an antibody Fab‐fragment
Author(s) -
Lilie Hauke,
Jaenicke Rainer,
Buchner Johannes
Publication year - 1995
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 - 10.1002/pro.5560040511
Subject(s) - chemistry , folding (dsp implementation) , contact order , denaturation (fissile materials) , phi value analysis , protein folding , downhill folding , crystallography , biophysics , intramolecular force , guanidinium chloride , complementarity determining region , stereochemistry , native state , biochemistry , peptide sequence , biology , enzyme , gene , electrical engineering , nuclear chemistry , engineering
Abstract Antibody folding is a complex process comprising folding and association reactions. Although it is usually difficult to characterize kinetic folding intermediates, in the case of the antibody Fab fragment, domain‐domain interactions lead to a rate‐limiting step of folding, thus accumulating folding intermediates at a late step of folding. Here, we analyzed a late folding intermediate of the Fab fragment of the monoclonal antibody MAK 33 from mouse ( k /IgGl). As a strategy for accumulation of this intermediate we used partial denaturation of the native Fab by guanidinium chloride. This denaturation intermediate, which can be populated to about 90%, is indistinguishable from a late‐folding intermediate with respect to denaturation and renaturation kinetics. The spectroscopic analysis reveals a native‐like secondary structure of this intermediate with aromatic side chains only slightly more solvent exposed than in the native state. The respective partner domains are weakly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a nativelike structure, follows a two‐step mechanism. In this mechanism, presumably hydrophobic interactions are followed by rearrangements leading to the exact complementarity of the contact sites of the respective domains.