Lanthanide-binding helix-turn-helix peptides: solution structure of a designed metallonuclease.
A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in alpha-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 +/- 0.3 microM) and for Eu(III) (6.2 +/- 0.3 microM) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helix-turn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metal-binding loop but also retains the alpha-alpha corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence.
Welch
Joel T
JT
Department of Chemistry and College of Medicine NMR Facility, University of Iowa, Iowa City, IA 52242, USA.
Kearney
William R
WR
Franklin
Sonya J
SJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004950:Esterases / Q000737:chemistry*; D019077:Helix-Turn-Helix Motifs; D007700:Kinetics; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008667:Metalloproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D012996:Solutions
D028581:Lanthanoid Series Elements; D008667:Metalloproteins; D010455:Peptides; D012996:Solutions; D004950:Esterases