Parallel pathways in the folding of a short-term denatured scFv fragment of an antibody.
BACKGROUND
Antibodies are prototypes of multimeric proteins and consist of structurally similar domains. The two variable domains of an antibody (VH and VL) interact through a large hydrophobic interface and can be expressed as covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv fragments after long-term denaturation in guanidinium chloride is known to be slow. In order to delineate the nature of the rate-limiting step, the folding of the scFv fragment of an antibody after short-term denaturation has been investigated.


RESULTS
Secondary structure formation, measured by H/D-exchange protection, of a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium chloride was shown to be multiphasic. NMR analysis shows that an intermediate with significant proton protection is observed within the dead time of the manual mixing experiments. Subsequently, the folding reaction proceeds via a biphasic reaction and mass spectrometry analyses of the exchange experiments confirm the existence of two parallel pathways. In the presence of cyclophilin, however, the faster of the two phases vanishes (when followed by intrinsic tryptophan fluorescence), while the slower phase is not significantly enhanced by equimolar cyclophilin.


CONCLUSIONS
The formation of an early intermediate, which shows amide-proton exchange protection, is independent of proline isomerization. Subsequently, a proline cis-trans isomerization reaction in the rapidly formed intermediate, producing 'non-native' isomers, competes with the fast formation of native species. Interface formation in a folding intermediate of the scFv fragment is proposed to prevent the back-isomerization of these prolines from being efficiently catalyzed by cyclophilin.
Freund
C
C
Department of Biochemistry, University of Zürich, Switzerland.
Gehrig
P
P
Baici
A
A
Holak
T A
TA
Plückthun
A
A
D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009154:Mutation; D019696:Peptidylprolyl Isomerase / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv; D019696:Peptidylprolyl Isomerase