Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies

Abstract

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, DjavadiOhaniance 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.

Antibodies as specific chaperones

“…Moreover, conformation sensitive antibodies against yeast cytochrome c, recognizing the native epitope at early stages of folding, did not change the slow kinetic phase of folding [21], and antibodies recognizing the folding inter mediate of hen lysozyme had no effect on restoration of catalytic activity during refolding [20]. Antibodies recog nizing a partially folded monomer of phage P22 tailspike protein did not influence the refolding yield of the protein from urea, while antibodies against the native trimer form of phage P22 tailspike protein inhibited trimerization [37]. Evidently, there is no universal mechanism of the effect of antibodies on folding; it is rather dependent on the epitope properties and the folding pathway of each particular protein.…”

Cataract as a Protein‐Aggregation Disease

“…In the best studied cases, inclusion bodies are formed from the polymerization of partially folded intermediates [14,159,160]. The irreversible association of protein chain within inclusion bodies is not due to disulfide bonds but rather to the same intimate interactions that keep native proteins folded [22,160,161]. This model implies that some stress on the crystallins generates partially unfolded species or otherwise conformational altered species, and these polymerize into the cataract.…”

Pressure treatment of tailspike aggregates rapidly produces on‐pathway folding intermediates

“…Tailspike protein aggregation involves specific interaction between chains, as mixing of denatured tailspike with other aggregation-prone proteins does not yield mixed aggregate species (Speed et al, 1996). Two possible mechanisms may drive tailspike aggregation: one model proposes that misfolding of the ␤-helix and misaligned ␤-sheet formation create sites susceptible to incorrect ␤-strand association; an alternative model reasons that interdigitated ␤-helices form with native-like strand alignment (Speed et al, 1997a).…”