De Novo Design and Structural Characterization of Proteins and Metalloproteins

DeGrado, William F.; Summa, Christopher M.; Pavone, Vincenzo; Nastri, Flavia; Lombardi, Angela

doi:10.1146/annurev.biochem.68.1.779

Cited by 564 publications

(560 citation statements)

References 308 publications

(336 reference statements)

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“…Finally, helix-stabilizing polar residues were placed at the most exposed positions to provide water solubility. An idealized ␥-␣ L -␤ interhelical loop (8,75) was included between the two pairs of helices. The resulting protein is designated ''Due Ferro 1'' (DF1), which means ''two iron'' in Italian.…”

Section: Resultsmentioning

confidence: 99%

“…Their methods instead begin with a fixed backbone structure (generally of a natural protein) and then search for convenient locations for introducing a metal ion-binding site. Over the last decade, these programs have been used successfully to graft a variety of structural and redox-active metal-binding sites into several different natural and designed proteins (6,8,17). However, because crystallographic or NMR structures are not available for any of these grafted sites, it is somewhat difficult to assess the merits of these methods relative to the de novo design approach described here.…”

Section: Discussionmentioning

confidence: 99%

“…The study of large proteins is hampered by their extreme complexity, and it is difficult to synthesize small-molecule models capable of simultaneously binding diiron, oxygen, and various substrates. Recently, we and others have sought a molecular middle ground between these two extremes through the design of small proteins and peptides that self assemble to form complexes with hemes and metal ions (5)(6)(7)(8).…”

mentioning

confidence: 99%

“…Further, peptide models address not only the issue of how the arrangement of atoms within an active site leads to function but also the very important question of how an amino acid sequence dictates the tertiary structure that supports this active site. Thus, several groups have described the design of minimal mimics of metalloproteins and heme-binding proteins that distill the quintessential elements believed to be responsible for the activities of metalloproteins into model proteins that are simpler and hence more easily understood than natural proteins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Through a careful characterization of the properties of such minimal metalloproteins, it should be possible to discern the features required for selective recognition of metal ions and for tuning their chemical properties.…”

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confidence: 99%

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Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Lombardi

Summa

Geremia

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 Å rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the GluXxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 Å. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation. P roteins use a limited repertoire of metal ion cofactors to help catalyze a multitude of reactions. For example, diiron sites (1-4) mediate reversible oxygen binding in hemerythrins, whereas they function as hydrolytic centers in phosphatases. Structurally similar diiron sites also mediate a number of oxygendependent oxidative processes. Ferritins serve as ferroxidases, while other diiron proteins catalyze hydroxylation, epoxidation, and desaturation reactions. Further, a diiron site in Escherichia coli ribonucleotide reductase is responsible for the formation of a Tyr radical. How do the structures of these proteins tune the chemical properties of a common diiron center to obtain such a diversity of highly specific catalysts? This question is being addressed through the study of the natural proteins as well as the study of small-molecule diiron complexes (1-4). Although impressive progress has been made on both fronts, these approaches have inherent limitations. The study of large proteins is hampered by their extreme complexity, and it is difficult to synthesize small-molecule models capable of simultaneously binding diiron, oxygen, and various substrates. Recently, we and others have sought a molecular middle ground between these two extremes through the design of small proteins and peptid...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Lombardi

Summa

Geremia

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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233

279

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“…62 Two frequently occurring three-residue loops, helix-(α L -β-β)-helix and helix-(γ-α L -β)-helix, are comprised of two common helical motifs. 10,62 First, the Ccapping Schellman motif (helix-α L -β) caps the end of the first helix. 63 Second, the Ncapping motif (β-helix) or hydrophobic staple (β-β-helix) caps the start of the second helix.…”

Section: Determinants Of the Free Energy Gapmentioning

confidence: 99%

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

et al. 2000

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De novo protein design has proven to be a powerful tool for understanding protein folding, structure, and function. In this Account, we highlight aspects of our research on the design of dimeric, four-helix bundles. Dimeric, four-helix bundles are found throughout nature, and the history of their design in our laboratory illustrates our hierarchic approach to protein design. This approach has been successfully applied to create a completely native-like protein. Structural and mutational analysis allowed us to explore the determinants of native protein structure. These determinants were then applied to the design of a dinuclear metal-binding protein that can now serve as a model for this important class of proteins.Protein folding is an important and multifaceted scientific problem. 1-8 One facet of this problem involves the prediction of three-dimensional structure from amino acid sequence, an endeavor the importance of which has been highlighted by the recent release of gene sequences of many whole organisms. As the size of the protein structural database expands, it will be increasingly possible to assign amino acid sequences to a given structural family on the basis of the homology of their sequences with proteins of known structure. Another facet of the folding problem is the delineation of the kinetic mechanism by which an unfolded protein chain undergoes the transition from a random coil with an astronomical number of configurations to a uniquely folded structure. A final facet of the protein folding problem is to understand, at the atomic level, the detailed physicochemical features that kinetically and thermodynamically direct the formation of an unique, cooperatively folded, native structure. This latter endeavor is particularly important as it lays the groundwork for the design of novel inhibitors of natural proteins as well as the construction of novel proteins and related biopolymers not found in nature.De novo protein design is an approach to the protein folding problem that critically tests our understanding of all these facets of this problem. 9,10 This method involves the design of a sequence that is intended to fold into a predetermined three-dimensional structure without the sequence being patterned after any natural protein. Through an iterative process of design and rigorous characterization, the principles governing protein folding and function can be evaluated. Thus, "failures" highlight gaps in our understanding, whereas "successes" confirm the principles used in the design and often provide simple model systems to further

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Metalloprotein Design & Engineering

2005

Encyclopedia of Inorganic Chemistry

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This review covers recent advances in metalloprotein design, with focus on different approaches to the design. Impressive progress has been made in designing metal‐binding sites in peptides, de novo designed proteins, and native protein scaffolds. The approach can be rational or combinatorial. Under rational design, redesigning an existing metal‐binding site to a new site with dramatically different structure and function complements well the design of new metal‐binding sites by revealing the role of specific residues responsible for a particular structural or functional feature of the metal‐binding site of interest. To create a new metal‐binding site, several approaches have been used, including design based on structural homology, by inspection, using automated computer search algorithms, or combination of the above approaches. In addition, modular approach by transplanting a conserved structural unit from one protein into another has also been shown to be effective. Design through combinatorial and evolution methods has also been successful as it requires little prior knowledge of the protein structure. Finally, introducing unnatural amino acids or nonnative metal ions/prosthetic groups to expand the repertoires of metalloproteins have been demonstrated. Successful examples of each of the approaches are given; advantages and disadvantages of the approaches are discussed; the outlook for future research is also presented.

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De Novo Design and Structural Characterization of Proteins and Metalloproteins

Cited by 564 publications

References 308 publications

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

Metalloprotein Design & Engineering

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