De novo design of a copper(II)-binding helix-turn-helix chimera: the prion octarepeat motif in a new context.
A chimeric Cu-binding peptide has been designed on the basis of a turn substitution of the prion (PrP) octarepeat Cu-binding site into the engrailed homeodomain helix-turn-helix motif (HTH). This system is a model for the investigation of a single PrP Cu-binding site in a defined protein context. The 28-mer Cu-HTH peptide P7 spectroscopically mimics the PrP octarepeat (P7 = TERRRQQLSHGGGWGEAQIKIWFQNKRA). The Cu(II)-binding affinity of P7 was determined by ESI-MS and tryptophan fluorescence titrations to be K(d) = 2.5 +/- 0.7 microM at pH = 7.0. The quenching of fluorescence of the Trp within the binding loop (underlined above) is pH dependent and highly specific for Cu(II). No Trp quenching was observed in the presence of divalent Zn, Mn, Co, Ni, or Ca ions, and ESI-MS titrations confirmed that these divalent ions do not appreciably bind to P7. The EPR spectrum of Cu(II)-P7 shows that the Cu environment is axial and consistent with 6-coordinate N(3)O(H(2)O)(2) or N(4)(H(2)O)(2) coordination (A( parallel) = 172 x10(-)(4) cm(-)(1); g( parallel) = 2.27), very similar to that of the PrP octarepeat itself. Also like PrP, circular dichroism studies show that apo P7 is predominantly disordered in solution, and the structure is slightly enhanced by Cu binding. These data show the Cu-PrP HTH peptide reproduces the Cu-binding behavior of a single PrP octarepeat in a new context.
Shields
S Brookhart
SB
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Franklin
Sonya J
SJ
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D008969:Molecular Sequence Data; D011328:Prions / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D011328:Prions; D011993:Recombinant Fusion Proteins; D003300:Copper