Abstract
Three peptide analogs of the helix-loop-helix Ca2+ binding unit, 21-, 26-, and 34-residues in length, similar in sequence to rabbit skeletal troponin C site III have been prepared by the solid-phase method. The CD spectra of the 21-residue fragment indicated very little secondary structure in aqueous medium in the absence of Ca2+. Addition of Ca2+ increased the secondary structure of the peptide but the KCa was very weak, 3.1 x 10(2) M-1. The same peptide in hydrophobic medium in the absence of Ca2+ had considerable secondary structure and the KCa value increased considerably, 3.5 x 10(5) M-1. The 26-residue peptide, containing 5 more residues on the NH2 terminus of the 21-residue peptide, showed slightly more secondary structure in aqueous medium in the absence of Ca2+. Addition of Ca2+ to this peptide raised the amount of secondary structure in the metal ion-peptide complex and resulted in a higher KCa value, 3.8 x 10(4) M-1. By assuming that the COOH-terminal region of the 26-residue peptide-metal ion complex assumes a structure similar to that of the 21-residue peptide-metal ion complex, one is able to assign the increase in structure to the NH2-terminal side of the Ca2+-binding loop. Hydrophobic medium further increased the secondary structure of this peptide and also increased the KCa value to 4.5 x 10(5) M-1, a value similar to that obtained for the 21-residue peptide. The 34-residue peptide contained a further 8 amino acid residues on the NH2 terminus of the 26-residue peptide. This peptide had considerable secondary structure in aqueous medium which increased in the presence of Ca2+. The peptide has a reasonable affinity for Ca2+ in aqueous medium, KCa = 2.6 x 10(5) M-1. Again, a hydrophobic medium increased both the amount of secondary structure and the Ca2+ affinity constant, KCa = 9.2 x 10(5) M-1. A model of Ca2+-induced folding of the three peptides under different conditions is described and results obtained from this model are used to describe Ca2+ binding to the four Ca2+ binding units in rabbit skeletal troponin C.
Highlights
Three peptide analogs of the helix-loop-helix Ca2' binding units in these proteins appear tobe similar, i.e. helixbinding unit, 21,26, and 34-residues in length, similar in sequence to rabbit skeletal troponin C site 111have been prepared by the solid-phasemethod
The Ca2+ binding helix-loop-helix conformation has been observed in the crystal structureof carp MCBP 4.25 (1) and predicted for the troponinC's ( 2 ),calrnodulins ( 3, 4 ),and light chains of myosin (5) through sequence homology with carp MCBP 4.25
Since the binding of Ca" to these proteins is accompanied by an increasein (Y helicity (6), it has been assumed that partof the helical regions of the helix-loop-helix Ca2+binding unit are induced by Cap+ and that this conformational change is responsible for the Ca'+-induced activity of these proteins
Summary
Three peptide analogs of the helix-loop-helix Ca2' binding units in these proteins appear tobe similar, i.e. helixbinding unit, 21-,26-, and 34-residues in length, similar in sequence to rabbit skeletal troponin C site 111have been prepared by the solid-phasemethod. Eachindividual amino acid residue in the Ca"+ binding unit can contributetothe Ca"-induced activity via conformation effectsthrough aninfluence on loop and helix formation or complexation effects through aninfluence on the side chain interaction with the Ca2+cation or increase in structure to the NH2-terminalside of the both.
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