The scFv fragment of the antibody hu4D5-8: evidence for early premature domain interaction in refolding.
Fluorescence spectroscopy and 1H/2H-exchange techniques have been applied to characterize the folding of an scFv fragment, derived from the humanized anti-HER2 antibody hu4D5-8. A stable intermediate, consisting of a native VL domain and an unfolded VH domain, is populated under equilibrium unfolding conditions. A partially structured intermediate, with 1H/2H-exchange protection significantly less than that of the two isolated domains together, is detectable upon refolding the equilibrium-denatured scFv fragment. This means that the domains in the heterodimer do not fold independently. Rather, they associate prematurely before full 1H/2H-exchange protection can be gained. The formation of the native heterodimer from the non-native intermediate is a slow, cooperative process, which is rate-limited by proline cis/trans-isomerization. Unproductive domain association is also detectable after short-term denaturation, i.e. with the proline residues in native conformation. Only a fraction of the short-term denatured protein folds into the native protein in a fast, proline-independent reaction, because of spontaneous proline cis/trans-reisomerization in the early non-native intermediate. The comparison with the previously studied antibody McPC603 has now allowed us to delineate similarities in the refolding pathway of scFv fragments.
Jäger
M
M
Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057, Zürich, Switzerland.
Gehrig
P
P
Plückthun
A
A
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007147:Immunoglobulin Light Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D006859:Hydrogen; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011392:Proline; D018719:Erb-b2 Receptor Tyrosine Kinases