Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.
The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.
Speed
M A
MA
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA.
Morshead
T
T
Wang
D I
DI
King
J
J
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D017100:Bacteriophage P22 / Q000737:chemistry*; D015153:Blotting, Western; D000939:Epitopes / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry*; D015335:Molecular Probes; D017510:Protein Folding; D017376:Viral Tail Proteins / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015335:Molecular Probes; D017376:Viral Tail Proteins; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage