Chimeric HTH motifs based on EF-hands.
The design of a new peptide construct from two structurally equivalent basis motifs is reported. A chimera was designed from the helical regions of a helix-turn-helix (HTH) domain, incorporating the consensus EF-hand Ca-binding loop at the turn. Two 33-residue peptides were constructed: one (P3, designed) includes the 12-residue consensus EF-hand loop, while the other (P2, control) contains the reversed EF-hand loop sequence. The Eu(III) and Ca(II) binding properties of P2 and P3 were investigated by circular dichroism and NMR. The designed peptide (P3) is 25% helical in its Eu(III)-saturated form, and 14% helical with excess Ca(II). Both the free and Eu-bound peptides have inherent solution structure, as demonstrated by the helicity induced by the addition of trifluoroethanol solvent. While Eu(III) binding stabilizes the structure of P3, it destabilizes the structure of P2. The NMR titration of P3 with Eu(III) resulted in new resonances characteristic of Ca-bound EF-hand loops. As observed for isolated EF-hands, the resonances appear within the first 0.5 equivalents of Eu(III) added, suggesting that one metal ion organizes two equivalents of peptide to fold into the back-to-back dimer structure of native EF-hands. The EuP3 chimera, but not EuP2, has significant affinity for supercoiled plasmid DNA, causing a gel shift at concentrations as low as 10 microM EuP3 (50 microM base pairs). These results show our chimeric peptide combines the characteristics of the parent motifs, maintaining both metal binding and DNA affinity.
Kim
Y
Y
Department of Chemistry, University of Iowa, Iowa City 52242, USA.
Welch
J T
JT
Lindstrom
K M
KM
Franklin
S J
SJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry; D015195:Drug Design; D009682:Magnetic Resonance Spectroscopy; D008674:Metals, Rare Earth / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000737:chemistry
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D008674:Metals, Rare Earth; D010455:Peptides; D011993:Recombinant Fusion Proteins