Miniaturized metalloproteins: Application to iron–sulfur proteins Angela Lombardi, Daniela Marasco, Ornella Maglio*, Luigi Di Costanzo, Flavia Nastri*, and Vincenzo Pavone† Department of Chemistry, University of Napoli Federico II, Via Mezzocannone 4, I-80134 Napoli, Italy Communicated by William F. DeGrado, University of Pennsylvania School of Medicine, Philadelphia, PA, June 22, 2000 (received for review March 27, 2000) The miniaturization process applied to rubredoxins generated a parts. Several strategies can be conceived to obtain a miniatur- class of peptide-based metalloprotein models, named METP (min- ized model. Two or more parts could spontaneously associate to iaturized electron transfer protein). The crystal structure of Desul- give folded noncovalent self-assembled oligomers, or two or fovibrio vulgaris rubredoxin was selected as a template for the more parts could be covalently connected to give folded mono- ␥ construction of a tetrahedral (S -Cys)4 iron-binding site. Analysis of mers (7–10). We described the latter strategy in the design of the structure showed that a sphere of 17 Å in diameter, centered mimochromes, covalent helix-heme-helix sandwiches (11–14), on the metal, circumscribes two unconnected approximately C2 and we described the former in the design of DF1, a small ␤ symmetry related -hairpins, each containing the -Cys-(Aaa)2-Cys- four-helix bundle protein with an oxo-di-iron site in its interior sequence. These observations provided a starting point for the (15, 16). Mimochromes and DF1 have a common characteristic: design of an undecapeptide, which self assembles in the presence they are both C symmetric dimers, intended to reproduce the of tetrahedrally coordinating metal ions. The METP peptide was 2 quasi-symmetrical structure of a metalloprotein. The use of C synthesized in good yield by standard methodologies. Successful 2 symmetry is particularly advantageous because it simplifies the assembly of the METP peptide with Co(II), Zn(II), Fe(II͞III), in the expected 2:1 stoichiometry, was proven by UV-visible and circular design and reduces the size of the molecules to be synthesized dichroism spectroscopies. UV-visible analysis of the metal com- and may simplify their structural characterization. However, the plexes indicated the four Cys ligands tetrahedrally arrange around use of a C2 symmetry axis to construct a symmetric dimer self the metal ion, as designed. Circular dichroism measurements on assembled around metal ions leaves open the problem of pos- ⌳ ⌬ both the free and metal-bound forms revealed that the metal sible diastereomeric forms. and isomers of Co(III)- coordination drives the peptide chain to fold into a turned con- mimochrome I were unpredicted but experimentally observed formation. NMR characterization of the Zn(II)-METP complex fully (12). DF1 was instead designed to self assemble as a trans supported the structure of the designed model. These results prove antiparallel dimer, thus inhibiting the formation of a second that METP reproduces the main features of rubredoxin. diastereomeric form. Further, the design of noncovalent asym- metric dimers is complicated by homodimer formation. iniaturized proteins are peptide-based synthetic models of This paper describes a design concept as applied to Rds (rubre- natural macromolecular systems. They contain a minimum doxins), the simplest iron–sulfur proteins. Rds are small proteins M ͞ ␥ set of constituents necessary for an accurate reconstruction of with a Fe(II) Fe(III) high spin ion in a tetrahedral (S -Cys)4 site, defined structures and for a fine-tuned reproduction of defined which seem to participate in electron transfer (17). They display functions (1, 2). Their intermediate size between low molecular high similarity in both their three-dimensional structures and weight compounds and protein mutants makes miniaturized pro- metal-binding sequence, constituted by two pairs of Cys residues teins suitable for structure–function relationship studies: they are spaced as [-Cys-(Aaa)2-Cys-(Aaa)n-Cys-(Aaa)2-Cys-] (18–22). simple enough to avoid ambiguities of interpretation associated Several investigations are being carried out on both natural Rds with large proteins, and they provide sufficient size and chemical (17–23) and their mutants (24–32) and on model systems (33–39) diversity to allow the construction of active sites. to identify the factors tuning the properties of the active site. The miniaturization process can be rationally organized, once We report here the design, synthesis, and characterization of a a structural knowledge of the parent natural system to be prototype miniaturized Rd named METP (miniaturized electron miniaturized is available. It is necessary to define (i) the type and transfer protein). The coordination properties toward metal ions number of constituents to be assembled, (ii) the structure to be other than iron (zinc and cobalt) have also been examined to prove reconstructed, and (iii) the function to be reproduced. These the versatility of METP in reproducing the structure and properties aspects are strictly related. A larger number of constituents of other proteins with tetrahedral metal-binding sites. might be necessary for a more precise three-dimensional recon- struction and for a more specialized function. Materials and Methods Metalloproteins are well suited to miniaturization. The metal Molecular Modeling. A Silicon Graphics Indigo 2 workstation with center represents a pivotal point where spheres of variable diam- ͞ eters that circumscribe part of the protein are centered: the larger the INSIGHT II DISCOVER program package (Biosym Technologies, San Diego) was used. The consistent valence force field (CVFF) the diameter of the sphere, the larger the number of constituents ␥ included in the model. Comparative studies, in terms of structure with suitable parameters for the Fe(II)-(S -Cys)4 moiety (28) and and function, of a set of protein models, defined by spheres of with a distance-dependent dielectric constant was used for energy variable diameters, will allow better understanding of how the minimization, performed with the Newton–Raphson algorithm protein matrix modulates the unique features of the metal center, including electronic, redox, and catalytic properties (3–5). These Abbreviations: METP, miniaturized electron transfer protein; UV-vis, UV-visible; CD, circular properties are imprinted by the primary coordination shell—the dichroism; Rds, rubredoxins. composition, the number, and the geometry of ligands (6). They are *Permanent address: Centro di Studio di Biocristallografia del C.N.R., Via Mezzocannone 4, also regulated by the secondary coordination shell—the immediate I-80134 Napoli, Italy. surroundings of the primary coordination sphere—and they are †To whom reprint requests should be addressed. E-mail: [email protected]. finely modulated by long-range electrostatic factors (6). The publication costs of this article were defrayed in part by page charge payment. This For a sphere of a given diameter around the metal center, the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. constituents therein circumscribed are in general unconnected §1734 solely to indicate this fact. 11922–11927 ͉ PNAS ͉ October 24, 2000 ͉ vol. 97 ͉ no. 22 Downloaded by guest on October 2, 2021 until convergence. The modeling of METP was based on the crystal where [ZnP2], [CoP2], [Co]f, [Zn]f are the concentrations of structure of Desulfovibrio vulgaris Rd (ref. 19; PDB ID code Zn(II) complex, Co(II) complex, free Co2ϩ, and free Zn2ϩ, Zn Co Zn ϭ 8RXN). respectively. Kdiss was determined from Kdiss and Kex: Kdiss Co ͞ (1͞2) Kdiss (Kex) . Peptide Synthesis and Purification. METP peptide was synthesized by using a MilliGen peptide synthesizer by standard Fmoc͞ UV-Visible (UV-vis) Spectroscopy. UV-vis spectra were recorded on tert-butyl (But) strategy on a NovaSyn PR500 resin (Novabiochem, a Perkin–Elmer Lambda 7 UV Spectrophotometer by using Switzerland). Cleavage from the resin and sidechain deprotection quartz cells of 1 cm path length. Wavelength scans were per- ͞ ͞ ͞ was achieved with ethanedithiol H2O triisopropylsilane formed at room temperature, from 200 to 700 nm, with a 60 Ϫ trifluoroacetic acid 0.25:0.25:0.1:9.5 (vol͞vol͞vol͞vol) for 2 h. Ex- nm⅐min 1 scan speed. Anaerobic spectra were recorded in traction and precipitation with ether gave the crude material (yield rubber-sealed cuvettes under nitrogen. 80%). It was reduced by 3-h incubation in aqueous solution at pH 8 and 10-fold excess DTT. Purification by preparative HPLC gave Circular Dichroism (CD) Spectroscopy. CD spectra were obtained at room temperature on a Jasco (Easton, MD) J-715 dichrograph. the desired pure product, as assayed by analytical HPLC and Ϫ matrix-assisted laser desorption ionization time-of-flight spectrom- Data were collected at 0.2-nm intervals with a 20 nm⅐min 1 scan etry [molecular weight: calculated 1,151.4 atomic mass units (amu); speed, a 2-nm bandwidth, and a 16-s response, from 260 to 190 nm, ⅐ Ϫ1 obs 1152 Ϯ 1 amu]. and at 0.5-nm intervals with a 200 nm min scan speed, a 2-nm bandwidth, and a 2-s response from 250 to 800 nm; circular quartz Metal-Binding Experiments. All manipulations were performed un- cells of 0.01-cm and 1-cm path length were used for the far UV and der strictly anaerobic conditions. Both peptide and metal stock near UV-vis regions, respectively. Peptide concentrations were in solutions were freshly prepared. The concentration of stock METP the range 0.20–1.2 mM. CD intensities in the far UV and near UV-vis regions are expressed as mean residue ellipticities (degree peptide solutions was determined spectrophotometrically by using Ϫ Ϫ ␧ ϭ Ϯ ϫ 3 Ϫ1⅐ Ϫ1 2⅐ 1⅐ 1 230 1.52 ( 0.01) 10 M cm , as calculated from experi- cm dmol residue ) and total molar ellipticities, respectively. mentally determined Lambert and Beer’s law. The Co(II) and Zn(II) complexes were prepared by addition NMR Spectroscopy.