The de novo design of a rubredoxin‐like fe site

Abstract

A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B 1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [ F e ( S -c~s )~] ",-'. The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(I1) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(I1)-bound forms of the variant are folded correctly and are stable. The Fe(II1) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(II1) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.Keywords: de novo design; metalloprotein; protein engineering; rubredoxin Protein engineering and de novo design approaches have begun to afford a more detailed understanding of the balance of the forces that determine protein structure and function (Regan & DeGrado, 1988;Regan, 1993;Bryson et al., 1995;Regan, 1995;Dalal et al., 1997;Smith & Regan, 1997;Hellinga, 1998). To date, there are just a few examples of designed proteins that adopt a specified fold, and number which display novel activities is still smaller (Dill, 1990; Schafmeister et al., 1993;Jackson et al., 1994;Quinn et al., 1994;Yan & Erickson, 1994;Baltzer et al., 1996;Dahiyat & Mayo, 1997;Hill & DeGrado, 1998;QuCnCneur et al., 1998). A particular focus of functional design efforts has been the engineering of novel metal-binding sites. Metal-binding sites represent an attractive target because they play a variety of functions from stabilization of protein structure to catalytic roles such as the activation of water for nucleophilic attack, electron transfer, and redox activity. More recently, several groups have focused strongly on the design of metal-binding sites with particular activities in mind. For example, two metal-binding histidine residues have been engineered into rat trypsin to "switch" the catalytic activity on or off by disrupting the catalytic triad. The protein can be turned off by Cu(I1) chelation in the designed site and reactivated upon removal of Cu(I1) with EDTA (Halfon & Craik, 1996). In another design, a metal-binding site, based on the metal center of carbonic

Miniaturized metalloproteins: Application to iron–sulfur proteins

“…The intensities of the d-d transitions in the visible region are characteristic of Co(II) coordination in a tetrahedral geometry (47) and exclude higher coordination numbers. The intense ligand-to-metal charge transfer (LMCT) band at 350 nm indicates thiolate ligation, and its intensity is in good agreement with those observed in proteins and model systems (30,31,38,39). The position of the absorption maxima of both d-d and LMCT transitions, which are sensitive to the identity of the ligands and move toward lower energies as the number of thiolate ligands increases, is consistent with a (S ␥ -Cys) 4 coordination environment (38)(39)(40)(41).…”

“…Several investigations are being carried out on both natural Rds (17)(18)(19)(20)(21)(22)(23) and their mutants (24)(25)(26)(27)(28)(29)(30)(31)(32) and on model systems (33)(34)(35)(36)(37)(38)(39) to identify the factors tuning the properties of the active site.…”

Purification and Biophysical Characterization of a New [2Fe-2S] Ferredoxin from Azotobacter vinelandii, a Putative [Fe-S] Cluster Assembly/Repair Protein

“…The possibility of having two rubredoxin-like iron centers was ruled out because FdIV contains only 6 cysteine residues per molecule, which cannot accommodate formation of two rubredoxin-like 1Fe centers. This is further supported by the observation that we could not detect any EPR signal with g values of 4.3 and 9.5, which arise from the 3/2 and 1/2 kramers' doublets, respectively, of an S ϭ 5/2 multiplet of rubredoxin in the dithionitereduced state (data not shown) (73)(74).…”

Metalloprotein Design & Engineering

“…195 The protein was capable of undergoing several successive cycles of air oxidation and reduction by β-mercaptoethanol. 195 In another study, the Metal-Search program was used to design the same mononuclear [Fe(Cys) 4 ] center into the B1 domain of the IgGbinding protein G. 194 From these studies, Farinas and Regan recognized a limitation of the Dezymer and Metal-Search programs. Both programs used the fixed backbone of protein coordinates from either X-ray or average NMR structures.…”