Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Peptide-dependent stimulation of the ATPase activity of the molecular chaperone BiP is the result of conversion of oligomers to active monomers.

The molecular chaperone BiP purified from bovine liver (bBiP) exhibits a low basal level of ATPase activity that can be stimulated 3-6-fold by synthetic peptides (Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390). By contrast, recombinant murine BiP (rBiP) purified to homogeneity following expression in Escherichia coli exhibits a higher basal level of ATPase activity and is much less stimulated by synthetic peptides. Nondenaturing gel electrophoresis showed that rBiP is predominantly monomeric, while bBiP exists in multiple forms probably corresponding to differentially modified monomeric, dimeric, and higher oligomeric species. Some, but not all, synthetic peptides cause conversion of the oligomeric and modified species of bBiP to a monomeric form. We propose that the peptide-dependent ATPase stimulation observed for BiP reflects the conversion of inactive oligomeric and/or modified species into active monomers.

Conformational change in the N-terminal domain of the Escherichia coli tryptophan synthase beta 2 subunit induced by its interactions with monoclonal antibodies.

A fluorescence energy transfer signal was used to follow conformational changes occurring in two types of protein-protein complexes. The first complex studied was the native-like beta 2 subunit of Escherichia coli tryptophan synthase reconstituted by reassembly of the N- and C-terminal proteolytic domains of the beta chain. The other complexes were formed by the association of the N-terminal fragment (F1) with a monoclonal antibody that recognizes the native dimeric protein; four such complexes, obtained with different antibodies that recognize four distinct antigenic sites on native beta 2, were investigated. It was shown that a structural readjustment, which the isolated F1 domain was unable to undergo alone, was imposed upon F1 by interdomain interactions. Furthermore, with three of the four antibodies studied, the same conformational change in F1 also occurred after formation of the F1-antibody complex. These results demonstrate that, through an "induced fit mechanism", antigen-antibody stereospecific assembly can force the polypeptide chain to adopt a structure more closely resembling the conformation it has in the native protein.

Kinetic characterization of early immunoreactive intermediates during the refolding of guanidine-unfolded Escherichia coli tryptophan synthase beta 2 subunits.

This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded beta 2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of beta, F1 (N-terminal; Mw = 29,000) and F2 (C-terminal; Mw = 12,000). It is shown that, while selected as being "specific" for the native protein, these antibodies are all able to recognize early folding intermediates. The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed. On the contrary, the rate constants of appearance of two "native-like" epitopes, carried by F1, could be determined during the folding of beta chains. The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s-1 at 12 degrees C. This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured beta chains. Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured beta chains, the same early folding events seem to be involved in the appearance of this epitope. The rate constant was found to be significantly smaller (k = 0.02 s-1 at 12 degrees C) for the appearance of the epitope to antibody 9. This shows that the regain of immunoreactivity is not concerted within the F1 domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Renaturation of guanidine-unfolded tryptophan synthase by multi-mixing stopped-flow dilution in D2O.

Guanidine hydrochloride (GdnHCl) at high concentrations, e.g. 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins. The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 50-100-fold in a 'renaturation buffer'. Fast kinetic studies, using a stopped-flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing. Secondly, the density differences existing between the concentrated GdnHCl solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped-flows. Here, it is shown that these difficulties can be overcome by using a multi-mixing stopped-flow to achieve 2 successive 7-fold dilutions and by using heavy water (D2O) to adjust the density of the renaturation buffer. This enabled us to study the appearance of a short-lived intermediate during the folding of the beta 2-subunit of Escherichia coli tryptophan synthase.