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De novo design and characterization of copper centers in synthetic four-helix-bundle proteins
Zitatschlüssel ISI:000167631400010
Autor Schnepf, R and Horth, P and Bill, E and Wieghardt, K and Hildebrandt, P and Haehnel, W
Seiten 2186-2195
Jahr 2001
ISSN 0002-7863
DOI 10.1021/ja001880k
Adresse 1155 16TH ST, NW, WASHINGTON, DC 20036 USA
Journal J. Am. Chem. Soc
Jahrgang 123
Nummer 10
Monat MAR 14
Verlag AMER CHEMICAL SOC
Zusammenfassung 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 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.
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