Folding mechanism of the CH2 antibody domain.
The immunoglobulin C(H)2 domain is a simple model system suitable for the study of the folding of all-beta-proteins. Its structure consists of two beta-sheets forming a greek-key beta-barrel, which is stabilized by an internal disulfide bridge located in the hydrophobic core. Crystal structures of various antibodies suggest that the C(H)2 domains of the two heavy chains interact with their sugar moieties and form a homodimer. Here, we show that the isolated, unglycosylated C(H)2 domain is a monomeric protein. Equilibrium unfolding was a two-state process, and the conformational stability is remarkably low compared to other antibody domains. Folding kinetics of C(H)2 were found to consist of several phases. The reactions could be mapped to three parallel pathways, two of which are generated by prolyl isomerizations in the unfolded state. The slowest folding reaction, which was observed only after long-term denaturation, could be catalyzed by a prolyl isomerase. The majority of the unfolded molecules, however, folded more rapidly, on a time-scale of minutes. Presumably, these molecules also have to undergo prolyl isomerization before reaching the native state. In addition, we detected a small number of fast-folding molecules in which all proline residues appear to be in the correct conformation. On both prolyl isomerization limited pathways, the formation of partly structured intermediates could be observed.
Feige
Matthias J
MJ
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
Walter
Stefan
S
Buchner
Johannes
J
D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D011392:Proline