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 NH2-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 microM. Thus, all experiments with the peptides were performed at a concentration of 1 microM. Reducing agents cause these helical peptides to form random coils. Fluorescence anisotropy measurements of fluorescein-labeled peptide with the exposed hydrophobic face yielded a Kd = approximately 106 microM 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.
Highlights
It has been accepted for some time that the amino acid sequence of a protein contains all the information to specify its tertiary conformation [1]
It is believed that aggregation is a result of the association of hydrophobic surfaces that are normally hidden in the core of a folded protein but are exposed in partially folded states
Molecular chaperones are a group of cellular proteins known to be mediators of in vivo folding
Summary
Reagents and Proteins—All reagents were analytical grade. 1,1ЈBis(4-anilino)napthalene-5,5Ј-disulfonic acid (bisANS) was obtained from Molecular Probes (Junction City, OR). The linear (reduced) apamin-rhodanese hybrid peptides are designated as APA08r, APA09r, and APA14r, with the lower case r denoting the linear form. A second set of NMR spectra were acquired after the addition of 10 l of 2 M DTT to allow the uncyclized (reduced) peptide to be studied. Anisotropy measurements on APA08 and APA09 peptides, each of which had Lys labeled with fluorescein, were performed with an excitation wavelength of 492 nm and emission wavelength at 519 nm. To a cuvette containing 50 mM Tris-HCl, pH 7.8, a given peptide was added to a concentration of 1 M, and the anisotropy values were recorded at 20 °C To this solution, cpn that had been dialyzed against 50 mM Tris-HCl, pH 7.8, was added to the cuvette to a final concentration of 10 M, and the anisotropy value was measured
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