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Abstract

Two projects are described that use synthetic peptides to study protein structure. The EF-hand Ca²⁺ binding protein calbindin D[sub 9k] was studied by synthesizing and characterizing peptides corresponding to each Ca²⁺ binding domain. The peptide called calb 1 corresponds to the site I binding domain and the peptide called calb 2 corresponds to site II. Circular dichroism (CD) spectra in collaboration with ΔV and calorimetry experiments determined by other groups indicated that calb 1 alone does not bind Ca²⁺. Calb 2 binds Ca²⁺ in a mechanism whereby one Ca²⁺ enables two peptides to be folded into a helical conformation in an apparent dimer. A 1:1 mixture of calb 1 and calb 2, called calb 1/2, binds two equivalents of Ca²⁺ into a helical conformation in an apparent heterodimer. The mechanism appears to be stepwise in which calb 2 binds the first Ca²⁺ and calb 1 binds the second Ca²⁺. This indicates a positive cooperative effect conferred by calb 2 onto calb 1. Our results are consistent with previous work on the intact calbindin D[sub 9k] indicating a strong dependence on favourable electrostatic interactions between Ca²⁺ and side chain ligands for high binding affinity, and positive cooperativity between the two binding domains. CD experiments of calb 2 and calb 1/2 in the presence of 0.1 M KC1 showed a smaller increase in helical content upon Ca²⁺ addition when compared to the absence of salt. The results in the presence of 0.1 M KCl corroborate the importance of electrostatic interactions in the Ca²⁺ binding affinity of the peptides. A peptide called aac-heme, which corresponds to helices F and G (residues 80 to 108) of the ά-chain of horse methemoglobin, was synthesized and characterized by CD spectroscopy. The sequence of aac-heme adopts a structure, called an ά,ά-corner in the intact protein, which has been hypothesized to be a protein folding initiator (Efimov, A. V . (1984) FEBS Lett. 166, 33-38). We tested Efimov's hypothesis by analysing the CD spectra of aac-heme in aqueous buffer and trifluoroethanol (TFE) solutions. Aac-heme is moderately helical in aqueous buffer and this helicity is concentration dependent. This indicates that aac-heme aggregates to compensate for the lack of tertiary interactions which would otherwise be present in the intact protein. Aac-heme is fully helical in 60% TFE solution suggesting that a hydrophobic environment enhances the helicity of this peptide. These observations are consistent with a folding mechanism for the native hemoglobin whereby a hydrophobic collapse occurs first, followed by the formation of secondary structure. The results suggest that the ά,ά-corner could well initiate the secondary structural formation in hemoglobin and that this initiation would be even more pronounced if it was preceded by a hydrophobic collapse of the unfolded hemoglobin. Thus, the ά,ά-corner may be integral in the folding pathway, as Efimov proposed.