Model Peptide Studies Demonstrate That Amphipathic Secondary Structures Can Be Recognized by the Chaperonin GroEL (cpn60)

Abstract

The molecular chaperone cpn60 binds many unfolded proteins and facilitates their proper folding. Synthetic peptides have been used to probe the question of how cpn60 might recognize such a diverse set of unfolded proteins. Three hybrid peptides were synthesized encompassing portions of the bee venom peptide, apamin, and the sequence KWLAESVRAGK from an amphipathic helix in the NH 2 -terminal region of bovine rhodanese. Two disulfides connecting cysteine residues hold the peptides in stable helical conformations with unobstructed faces oriented away from the disulfides. Peptides were designed to present either a hydrophobic or hydrophilic face of the amphipathic helix that is similar to the one near the amino terminus of rhodanese. Aggregation of these peptides was detected by measuring 1,1-bis(4-anilino)napthalene-5,5-disulfonic acid (bisANS) fluorescence at increasing peptide concentrations, and aggregation was not apparent below 2 M. Thus, all experiments with the peptides were performed at a concentration of 1 M. Reducing agents cause these helical peptides to form random coils. Fluorescence anisotropy measurements of fluorescein-labeled peptide with the exposed hydrophobic face yielded a K d ‫؍‬ ϳ106 M for binding to cpn60, whereas there was no detectable binding of the reduced form. The peptide with the exposed hydrophilic face did not bind to cpn60 in either the oxidized or reduced states. Fluorescence experiments utilizing bisANS as a probe showed that binding of the helical hydrophobic peptide could induce the exposure of hydrophobic surfaces on cpn60, whereas the same peptide in its random coil form had no effect. Thus, binding to cpn60 is favored by a secondary structure that organizes and exposes a hydrophobic surface, a feature found in amphipathic helices. Further, the binding of a hydrophobic surface to cpn60 can induce further exposure of complementary surfaces on cpn60 complexes, thus amplifying interactions available for target proteins.

GroEL Binds Artificial Proteins with Random Sequences

“…The same conclusion can be drawn from the studies using ␣-casein as a substrate protein and crystal structure of minichaperone in which a peptide with a roughly extended conformation occupied the polypeptide binding site of GroEL (17). It should be added, however, that this does not exclude the possibility that some peptides that interact weakly with GroEL adopt a conformation of ␣-helix when binding to GroEL (37)(38)(39)(40). It is our current view that the polypeptide binding site of GroEL may be flexible enough to accept an ␣-helix form of peptides that expose hydrophobic residues but that an extended form of peptides with hydrophobic residues should be a more favorite substrate.…”

“…Studies using a variety of proteins as a model of substrate protein have been carried out, but they have led to several different proposals on the GroELrecognizable structural features, ranging from a fully unfolded to native-like conformation (28 -36). Some proteins (short peptides) containing ␣-helices were reported to bind to GroEL (37)(38)(39)(40), although it was shown that an all or mainly ␤-sheet protein (Fab fragment, pepsin, and green fluorescent protein) could bind to GroEL (16,41,42). On the other hand, binding of GroEL to unfolded proteins without an ordered secondary structure, apocytochrome c (33) and ␣-casein (21), was demonstrated.…”

GroEL-Mediated Protein Folding: Making the Impossible, Possible

“…These regions were highly populated with hydrophobic amino acids, forming both hydrophobic and amphipathic -helices in the native state. More recent studies with small model peptides also support a key role for large stretches of hydrophobic surface in substrate protein recognition by GroEL (Brazil et al, 1997;Chen and Sigler, 1999;Preuss et al, 1999;. Complementary observations on the role of hydrophobic interactions have also been made with GroEL itself.…”

Synthesis and Characterization of a Peptide Identified as a Functional Element in αA-crystallin

“…These data confirm that the chaperone sites represent hydrophobic site(s). The observation of bis-ANS binding to a 19-amino acid linear peptide was surprising, because binding of bis-ANS or other fluorescent probes to only synthetic cyclical peptides has been reported earlier (33,34).…”