De novo design and characterization of copper centers in synthetic four-helix-bundle proteins.
The design and chemical synthesis of de novo metalloproteins on cellulose membranes with the structure of an antiparallel four-helix bundle is described. All possible combinations of three different sets of amphiphilic helices were assembled on cyclic peptide templates which were bound by a cleavable linker to the cellulose. In the hydrophobic interior, the four-helix bundle proteins carry a cysteine and several histidines at various positions for copper ligation. This approach was used successfully to synthesize, for the first time, copper proteins based on a four-helix bundle. UV-vis spectra monitored on the solid support showed ligation of copper(II) by about one-third out of the 96 synthesized proteins and tetrahedral complexes of cobalt(II) by most of these proteins. Three of the most stable copper-binding proteins were synthesized in solution and their structural properties analyzed by spectroscopic methods. Circular dichroism, one-dimensional NMR, and size-exclusion chromatography indicate a folding into a compact state containing a high degree of secondary structure with a reasonably ordered hydrophobic core. They displayed UV-vis absorption, resonance Raman, and EPR spectra intermediate between those of type 1 and type 2 copper centers. The present approach provides a sound basis for further optimizing the copper binding and its functional properties by using combinatorial protein chemistry guided by rational principles.
Schnepf
R
R
Contribution from the Albert-Ludwigs-Universität Freiburg, Institut für Biologie II/Biochemie, Schänzlestrasse 1, D-79104 Freiburg, Germany.
Hörth
P
P
Bill
E
E
Wieghardt
K
K
Hildebrandt
P
P
Haehnel
W
W
D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D011506:Proteins; D003300:Copper