title: Conformational studies on calcium binding by tBoc-Leu-Pro-Tyr-Ala-NHCH3, a tyrosine kinase substrate, in a nonpolar solvent.
abstract: With a view to understanding the structural requirement for tyrosine phosphorylation, we have examined the free and Ca(2+)-bound conformations of the synthetic peptide tBoc-Leu-Pro-Tyr-Ala-NHCH3, a substrate for a protein tyrosine kinase, using circular dichroism (CD), 1H and 13C nuclear magnetic resonance (NMR) and molecular modeling methods. CD spectrum of the free peptide in water showed a random coil structure, while the spectrum in acetonitrile was indicative of a folded structure containing a type III beta-turn. Dihedral angle data derived from JNH-CH coupling constants, as well as two-dimensional 1H-COSY and NOESY spectral analyses, showed that the peptide adopts a conformation close to the 3(10)-helix. Ca2+ binding by the peptide, as monitored by CD spectral changes, was quite weak in water. However, substantial CD spectral changes were observed in the peptide on addition of Ca2+ in acetonitrile suggestive of major conformational alterations due to Ca2+ binding. Analysis of the binding isotherms at 25 degrees C obtained from CD data in acetonitrile indicated a 2:1 peptide:Ca2+ ("sandwich") complex to be the dominant species with a Kd of about 30 microM. A 1:1 complex was also present and became significant at Ca2+:peptide ratios above 1. By comparison, the peptide formed a predominantly 1:1 complex with Mg2+ with a Kd of about 40 microM. 13C-NMR data showed that a mixture of cis and trans conformers (arising from rotation around the Leu-Pro bond) in the free peptide changes over to the all-trans form on coordination of the peptide carbonyl groups to the Ca2+ ion. 1H-NOESY data of the Ca2+ complex revealed several interactions involving the sidechains of two peptide molecules in the sandwich. Molecular modeling and energy minimization with and without the input of NOESY-derived distance constraints showed the sandwich complex to be an energetically very favourable conformation. Besides its relevance in terms of the possible involvement of divalent cations in substrate-tyrosine kinase interaction, the conformational characterization of tBoc-Leu-Pro-Tyr-Ala-NHCH3 and its Ca2+ complex should help understand the conformational determinants for Ca(2+)-binding by linear peptides.
authors.affiliation: Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
authors.forename: V S
authors.initials: VS
authors.lastname: Ananthanarayanan
authors.forename: A
authors.initials: A
authors.lastname: Saint-Jean
authors.forename: B V
authors.initials: BV
authors.lastname: Cheesman
authors.forename: D W
authors.initials: DW
authors.lastname: Hughes
authors.forename: A D
authors.initials: AD
authors.lastname: Bain
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; C086280:tert-butyloxycarbonyl-leucyl-prolyl-tyrosyl-alanyl-methylamide; D011505:Protein-Tyrosine Kinases; D002118:Calcium