Structure of α-α-hairpins with short connections

Ефимов, А. В.

doi:10.1093/protein/4.3.245

Cited by 80 publications

(65 citation 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%

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.

233

279

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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%

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.

233

279

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“…The helices within each monomer span residues 8-22 and 27-44 as defined by the pattern of hydrogen bonding as well as the backbone torsion angles and . The loop region comprises residues 24-26 and is characterized by a ␥␣ L ␤ conformation (7,38). In this turn, the first helix is capped in a conformation that lies between an ␣ L and a Schellman motif (39) in which Lys-25 adopts a left-handed helix conformation.…”

Section: Resultsmentioning

confidence: 99%

Preorganization of molecular binding sites in designed diiron proteins

Maglio

Nastri²,

Pavone³

et al. 2003

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

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De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (Kdiss ‫؍‬ 0.001 fM), but the dissociation constant increases to 0.6 nM (⌬⌬G ‫؍‬ 5.4 kcal͞ mol monomer) as the active-site cavity is increased to accommodate small molecules.T he requirements for protein stability versus function are often diametrically opposed. The folded conformations of proteins are stabilized by maximizing the burial of hydrophobic groups, minimizing voids, and forming intramolecular hydrogen bonds (1, 2). In contrast, binding and enzymatic functions generally require active-site clefts replete with solventexposed hydrophobic groups and hydrogen-bonding groups, which are essential for proper binding of substrates. This tradeoff between conformational stability and function presents a particularly large challenge to protein design (3), forcing the designer to skirt the waters between Scylla and Charybdis.The stability͞function tradeoff is particularly apparent in metalloproteins. Structural metal-binding sites in proteins frequently achieve stability by binding metal ions in coordinately saturated ligand environments with idealized ligand-metal bond geometries (4, 5). However, functional sites in metalloenzymes frequently contain coordinately unsaturated metal ions that are positioned appropriately for binding substrates; they also sometimes display geometries not frequently observed in simple, small-molecule metal-ligand complexes (4, 5). Further, the active sites of metalloproteins are frequently preorganized in the absence of metal ions, which requires the burial of polar ligands at the expense of folding free energy (6). The preorganization imparts tight and geometrically specific metal binding by assuring that the protein imparts its own structural pr...

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“…In this descending hierarchy, supersecondary structure is comprised of adjacent, interacting elements of regular secondary structure (i.e., (YLY, /3p, or pap units). Such higher order units are common in proteins (Levitt & Chothia, 1976; in particular, see Efimov, 1991;Richardson & Richardson, 1988b).…”

Section: Capping Studies In Simulationsmentioning

confidence: 99%

Helix capping

1998

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Helix-capping motifs are specific patterns of hydrogen bonding and hydrophobic interactions found at or near the ends of helices in both proteins and peptides. In an a-helix, the first four >N-H groups and last four >C=O groups necessarily lack intrahelical hydrogen bonds. Instead, such groups are often capped by alternative hydrogen bond partners. This review enlarges our earlier hypothesis (Presta LG, Rose GD. 1988. Helix signals in proteins. Science 240:1632-1641) to include hydrophobic capping. A hydrophobic interaction that straddles the helix terminus is always associated with hydrogen-bonded capping. From a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix N-terminus and four at the C-terminus. The consensus sequence patterns of these seven motifs, together with results from simple molecular modeling, are used to formulate useful rules of thumb for helix termination. Finally, we examine the role of helix capping as a bridge linking the conformation of secondary structure to supersecondary structure.Keywords: alpha helix; protein folding; protein secondary structure The a-helix is characterized by consecutive, main-chain, i + i -4 hydrogen bonds between each amide hydrogen and a carbonyl oxygen from the adjacent helical turn (Pauling & Corey, 1951). This pattern (Fig. 1) is interrupted at helix termini because, upon termination, no turn of helix follows to provide additional hydrogen bond partners. Such end effects are substantial, encompassing two-thirds of the residues for the protein helix of average length (Presta & Rose, 1988). Further, helix geometry hinders solvent access to amide groups in the first turn of the helix, inhibiting interaction with water and necessitating alternative hydrogen bonds. The term helix "capping" (Richardson & Richardson, 1988a) has been used to describe such alternative hydrogen bond patterns that can satisfy backbone >N -H and >C = 0 groups in the initial and final turns of the helix (Presta & Rose, 1988).Many studies involving helix capping have been conducted since publication of our initial hypothesis nine years ago (Presta & Rose, 1988). As we had proposed, amide hydrogens at the helix N-terminus are indeed satisfied predominantly by side-chain H-bond acceptors. In contrast, carbonyl oxygens at the C-terminus are satisfied primarily by backbone >N -H groups from the turn following the helix. Further, these hydrogen-bonding patterns at either helix end are accompanied by a companion hydrophobic interaction between apolar residues in the a-helix and its flanking turn. This hydrophobic component of helix capping was unanticipated.The main purpose of this review is to enlarge our previous definition of helix capping and to document the common capping motifs. Qpically, protein helices terminate in a hydrophobic interaction that straddles the helix termini (i.e., an interaction between two hydrophobic residues close in sequence, one within the helix, the other external to the helix). In this interaction,...

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Structure of α-α-hairpins with short connections

Cited by 80 publications

References 0 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

Preorganization of molecular binding sites in designed diiron proteins

Helix capping

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