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J Am Chem Soc. 2001 Oct 24;123(42):10255-64.

Helix-loop-helix peptides as scaffolds for the construction of bridged metal assemblies in proteins: the spectroscopic A-cluster structure in carbon monoxide dehydrogenase.

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Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.


Four helix-loop-helix 63mer peptides were designed and synthesized in order to assess the utility of peptides as scaffolds for the stabilization of complex metal sites in proteins. Bridged assembly [Ni(II)-(mu(2)-S.Cys)-Fe(4)S(4)], consistent with spectroscopic information on the A-cluster of carbon monoxide dehydrogenase, was chosen as the target assembly. The peptides consist of two helices with approximately 20 residues connected by a flexible loop containing the ferredoxin consensus sequence Cys-Ile-Ala-Cys-Gly-Ala-Cys to bind the Fe(4)S(4) cluster. A fourth cysteine was positioned to serve as the bridging ligand between the cluster and Ni(II). Three other binding residues were incorporated in appropriate positions to constitute a binding site for Ni(II). One of the peptides was designed with an N(3)S (His(3)Cys) site, and each of the other three with N(2)S(2) (His(2)Cys(2)) sites. A detailed account of the synthesis and characterization of the peptides and their metalloderivatives is presented. The four peptides were synthesized using an Fmoc/t-Bu-based solid-phase strategy, purified by reversed-phase HPLC, and characterized by ES-MS. On the basis of size-exclusion chromatography and circular dichroism spectropolarimetry, these peptides appear to dimerize in solution to form four-helix bundles of high helical contents. Reactions of the peptides with preformed cluster [Fe(4)S(4)(SCH(2)CH(2)OH)(4)](2)(-) and subsequent purification by column chromatography yield a product consistent with the incorporation of one [Fe(4)S(4)](2+) cluster per 63mer, as judged from absorption and Mössbauer spectra. Addition of a Ni(II) salt to the [Fe(4)S(4)]-peptides results in an apparent equilibrium between free Ni(II) and a peptide-bound nickel form, as established by column chromatography studies. Nickel EXAFS data (Musgrave, K. B.; Laplaza, C. E.; Holm, R. H.; Hedman, B.; Hodgson, K. O. Results to be published.) provide strong evidence that the peptide-bound nickel binds in the desired site in two of the metallopeptides. This work represents the first exploration of peptides as scaffolds for the support of biologically relevant bridged assemblies containing iron-sulfur clusters.

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