CONFORMATIONAL STUDIES ON CALCIUM-BINDING BY TBOC-LEU-PRO-TYR-ALA-NHCH3, A TYROSINE KINASE SUBSTRATE, IN A NONPOLAR SOLVENT

With a view to understanding the structural requirement for tyrosine p hosphorylation, we have examined the free and Ca2+-bound conformations of the synthetic peptide tBoc-LeuPro-Tyr-Ala-NHCH3, a substrate for a protein tyrosine kinase, using circular dichroism (CD), H-1 and C-13 nuclear magnetic resonance (NMR) and molecular modeling methods. CD sp ectrum of the free peptide in water showed a random coil structure, wh ile the spectrum in acetonitrile was indicative of a folded structure containing a type III P-turn. Dihedral angle data derived from J(NH-CH ) coupling constants, as well as two-dimensional H-1-COSY and NOESY sp ectral analyses, showed that the peptide adopts a conformation close t o the 3(10)-helix. Ca2+ binding by the peptide, as monitored by CD spe ctral changes, was quite weak in water. However, substantial CD spectr al changes were observed in the peptide on addition of Ca2+ in acetoni trile suggestive of major conformational alterations due to Calf bindi ng. Analysis of the binding isotherms at 25 degrees C obtained from CD data in acetonitrile indicated a 2:1 peptide:Ca2+ (''sandwich'') comp lex to be the dominant species with a K-d of about 30 mu M. A 1:1 comp lex was also present and became significant at Ca2+:peptide ratios abo ve 1. By comparison, the peptide formed a predominantly 1:1 complex wi th Mg2+ with a K-d of about 40 mu M, C-13-NMR data showed that a mixtu re of cis and trans conformers (arising from rotation around the Leu-P ro bond) in the free peptide changes over to the all-trans form on coo rdination of the peptide carbonyl groups to the Ca2+ ion. H-1-NOESY da ta of the Ca2+ complex revealed several interactions involving the sid echains of two peptide molecules in the sandwich. Molecular modeling a nd energy minimization with and without the input of NOESY-derived dis tance constraints showed the sandwich complex to be an energetically v ery favourable conformation. Besides its relevance in terms of the pos sible involvement of divalent cations in substrate-tyrosine kinase int eraction, the conformational characterization of tBoc-Leu-Pro-Tyr-Ala- NHCH3 and its Ca2+ complex should help understand the conformational d eterminants for Ca2+-binding by linear peptides.