IN-VITRO AND RIBOSOME-BOUND FOLDING INTERMEDIATES OF P22 TAILSPIKE PROTEIN DETECTED WITH MONOCLONAL-ANTIBODIES

It remains unclear whether polypeptide chains renaturing in vitro from strong denaturants proceed through the same folding pathway as chains released from ribosomes within cells. Folding intermediates formed bo th in vivo and in vitro have been examined using three monoclonal anti bodies shown previously to recognize different epitopes of the native P22 tailspike protein (Friguet, B., Djavadi-Ohaniance, L., Haase-Petti ngell, C, A, King, J., and Goldberg, M. E. (1990) J. Biol. Chem. 265, 10347-10351). The tailspike protein was reconstituted from polypeptide chains unfolded by urea as described by Fuchs ct al. (Fuchs, A., Seid erer, C., and Seckler, R. (1991) Biochemistry 30, 6598-6604), and the appearance of immunoreactive forms during the refolding was monitored. The three antibodies discriminated intermediates at different stages in the folding pathway. On the basis of the reconstitution pathway det ermined from spectroscopic and hydrodynamic measurements by Fuchs ct a l. (1991), monoclonal antibody (mAb) 236-3 recognized partially folded monomers, mAb 155-3 recognized folded protomers in a protrimer specie s, and mAb 33-2 recognized the native trimer. The kinetics of appearan ce of the immunoreactive forms during the in vitro refolding of the pr otein in crude extracts of phage-infected cells was similar to that ob served with the pure tailspike. Thus, the antibodies provided probes f or the chain folding and association pathway in vivo. The conformation of the ribosome-bound tailspike polypeptide chains of the infected ce lls was analyzed with the three antibodies. The antibodies recognizing native trimer and the protrimer did not bind chains associated with t he ribosomes. Antibody 236-3, which recognized structured monomers in vitro, bound to the polypeptide chains still associated with ribosomes . This result suggests that steps that take place in solution during i n vitro refolding may occur in a ribosome-bound state in vivo.