The rate-limiting steps for the folding of an antibody scFv fragment.
The refolding kinetics of a single-chain Fv (scFv) fragment, derived from the phosphorylcholine binding antibody McPC603, was investigated. Both prolyl-peptide bonds which are cis in the native state affect the refolding kinetics of long-term denatured protein. The rate-limiting step is the trans --> cis isomerization at the ProL95-peptide bond, which is catalyzed by peptidyl-prolyl-cis/trans-isomerase (PPIase), and is the prerequisite for correct V(H)/V(L) domain association. Refolding of short-term denatured protein resulted in complex refolding kinetics, too. This kinetic heterogeneity could be ascribed to cis --> trans re-isomerization at the ProL95-peptide bond to the wrong conformation in a folding intermediate. PPIase was shown to increase the fraction of slowly folding species, thereby competing with the fast folding of short-term denatured scFv, having native proline conformations. A trapped intermediate is rapidly populated, and the return from this state becomes rate-limiting.
Jäger
M
M
Biochemisches Institut der Universität Zürich, Switzerland.
Plückthun
A
A
D000595:Amino Acid Sequence; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D017354:Point Mutation; D011392:Proline; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D011392:Proline; D019696:Peptidylprolyl Isomerase