Characterization of a quaternary-structured folding intermediate of an antibody Fab-fragment.
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 (kappa/IgG1). 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 weekly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a native-like 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.
Lilie
H
H
Institut für Biophysik & Physikalische Biochemie, Universität Regensburg, Germany.
Jaenicke
R
R
Buchner
J
J
D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006146:Guanidines / Q000494:pharmacology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
D000178:Acrylamides; D000911:Antibodies, Monoclonal; D004220:Disulfides; D006146:Guanidines; D007140:Immunoglobulin Fab Fragments; D014364:Tryptophan; D004229:Dithiothreitol