Solution structure and dynamics of a
            <i>de novo</i>
            designed three-helix bundle protein

Walsh, Scott T. R.; Cheng, Hong; Bryson, James W.; Röder, Heinrich; DeGrado, William F.

doi:10.1073/pnas.96.10.5486

Cited by 200 publications

(221 citation statements)

References 59 publications

(71 reference statements)

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“…The ground-breaking work of DeGrado and coworkers (17)(18)(19)(20) has led to the development of well packed helix bundle proteins with natural-like sequences and stabilizing interactions. In their work, and pioneering work from other laboratories, a specific fold has been achieved via the formation of tertiary hydrogen bonds (20)(21)(22)(23), crystal contacts (18,24), disulfide bonds (25,26) or favorable charge-charge interactions (19,20,23,24,26,27). Previous work toward simplifying proteins has shown that a native-like Src homology 3 fold can be achieved with chiefly five different residues (although 13 in total) (28), and a four-helix bundle protein can be formed from only nine different residues (29).…”

Section: The Presence Of An Aromatic Ring At the C Terminus Is Requirmentioning

confidence: 99%

Getting specificity from simplicity in putative proteins from the prebiotic Earth

Osa

Bateman

et al. 2007

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

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Can unique protein structures arise from a limited set of amino acids present on the prebiotic Earth? To address this question, we have determined the stability and structure of KIA7, a 20-residue polypeptide containing chiefly Lys, Ile, and Ala. NMR methods reveal that KIA7 tetramerizes and folds on the millisecond time scale to adopt a four-helix X-bundle structure with a tightly and specifically packed core. Denaturation studies and hydrogen exchange measurements of KIA7 and several variants demonstrate that ridges-into-grooves packing of Ala and Ile side chains and the packing of a C-terminal aromatic group into the hydrophobic core are sufficient to give rise to a rather stable, well folded protein structure, with no favorable electrostatic interactions or tertiary or quaternary hydrogen bonds. Both modern proteins and RNAs can adopt specific structures, but RNAs do so with a limited ''alphabet'' of residues and types of stabilizing interactions. The results reported here show that specific, well folded protein structures can also arise from a highly reduced set of stabilizing interactions and amino acids that are thought to have been present on the prebiotic Earth.four-helix bundle ͉ NMR spectroscopy ͉ protein stability ͉ chemical evolution ͉ protein folding I n the prebiotic Earth, hydrophobic and charged amino acids were thought to have been rather common, whereas polar amino acids were rare (1, 2). Could such a limited set of amino acids have given rise to unique well folded proteins? To gain insight into this question, we have studied the conformational stability and structure of KIA7, a 20-residue polypeptide containing chiefly Lys, Ile, and Ala, plus a -Gly-Gly-Tyr extension for concentration determination, which self-associates to form a well packed, four-helix bundle protein.KIA7 was previously identified from a combinatorial peptide library as having a highly helical structure (as gauged by CD), a well packed hydrophobic core (1-anilino-8-naphthalene sulfonate fluorescence), and a specific tetrameric quaternary structure (analytical ultracentrifugation) (3, 4). The positively charged lysine side chains prevent the formation of higherordered oligomers. Because no tertiary hydrogen bonds, higherorder oligomerization or crystal packing interactions, or favorable electrostatic interactions are present in KIA7, this study constitutes a test of whether or not a specific, unique structure can arise from a highly limited set of stabilizing interactions, namely, the hydrophobic effect and van der Waals interactions.In addition to KIA7, two other members of the KIA series of peptides, KIA5 and KIA10, were studied (Table 1). These peptides are variants of the KIA7 sequence and possess lesser degrees of ordered structure (3). A stabilized KIA7 variant with a disulfide cross-link, CG 3 KIA7, was also examined, and additional peptides truncating the -Gly-Gly-Tyr C-terminal extension, KIA7⌬, or carrying Tyr to Phe, KIA7F, or Tyr to Ile, KIA7I, substitutions were studied to probe the possible stabilizing or struc...

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Section: The Presence Of An Aromatic Ring At the C Terminus Is Requirmentioning

confidence: 99%

Getting specificity from simplicity in putative proteins from the prebiotic Earth

Osa

Bateman

et al. 2007

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

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“…Unfortunately, initial attempts to design proteins led to structures that formed molten globulelike states with dynamic behavior relative to natural proteins. More recently, it has been possible to design small uniquely folded proteins that incorporate all of the commonly occurring secondary structural and supersecondary structural motifs (29)(30)(31)(32)(33)(34)(35)(36). These studies illustrated the delicate interplay of forces that define the uniquely folded structures of proteins; hydrophobicity provides a strong driving force for folding, but designs based on this consideration alone often adopt dynamically averaging Abbreviations: DF1, Due Ferro 1; PDB, Protein Data Bank.…”

mentioning

confidence: 99%

“…Side-chain repacking algorithms search for combinations of amino acid side chains capable of packing together in efficient low-energy combinations. However, with a few exceptions (36,46), these methods have been used to redesign natural proteins rather than to engineer novel structures. Further, a major limitation of early side-chain repacking algorithms was their use of rigid backbone geometries, which placed somewhat artificial restrictions on the number of combinations of side chains that may be accommodated within a protein core.…”

mentioning

confidence: 99%

“…Therefore, methods for the design of uniquely folded cofactor-binding proteins are needed. Recent computational approaches to de novo protein design have allowed the synthesis of novel proteins that fold into conformationally unique structures (36,(43)(44)(45). Side-chain repacking algorithms search for combinations of amino acid side chains capable of packing together in efficient low-energy combinations.…”

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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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“…Both genetic algorithms and simulated annealing have been successfully applied to re-design a number of natural proteins: 434 Cro (Desjarlais & Handel, 1995), ubiquitin (Johnson et al, 1999), the B1 domain of protein G (Jiang et al, 2000), the WW domain (KraemerPecore et al, 2001), and helical bundles (Bryson et al, 1998;Jiang et al, 1997). In many cases, these methods have aided in identifying experimentally viable sequences (Kraemer-Pecore et al, 2001;Walsh et al, 1999). However, since stochastic search methods do not always identify global optima (Voigt et al, 2000), other search methods have also been developed.…”

Section: "Directed" Methods Of Protein Designmentioning

confidence: 99%

Progress in the development and application of computational methods for probabilistic protein design

Park

Kono²,

Wang

et al. 2005

Computers & Chemical Engineering

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. (2004). Progress in the development and application of computational methods for probabilistic protein design. Retrieved from http://repository.upenn.edu/cbe_papers/2 Progress in the development and application of computational methods for probabilistic protein design AbstractProteins exhibit a wide range of physical and chemical properties, including highly selective molecular recognition and catalysis, and are also key components in biological metabolic, catabolic, and signaling pathways. Given that proteins are well-structured and can now be rapidly synthesized, they are excellent targets for engineering of both molecular structure and biological function. Computational analysis of the protein design problem allows scientists to explore sequence space and systematically discover novel protein molecules. Nonetheless, the complexity of proteins, the subtlety of the determinants of folding, and the exponentially large number of possible sequences impede the search for peptide sequences compatible with a desired structure and function. Directed search algorithms, which identify directly a small number of sequences, have achieved some success in identifying sequences with desired structures and functions. Alternatively, one can adopt a probabilistic approach. Instead of a finite number of sequences, such calculations result in a probabilistic description of the sequence ensemble. In particular, by casting the formalism in the language of statistical mechanics, the site-specific amino acid probabilities of sequences compatible with a target structure may be readily identified. The computational probabilities are well suited for both de novo protein design of particular sequences as well as combinatorial, library-based protein engineering. The computed site-specific amino acid profile may be converted to a nucleotide base distribution to allow assembly of a partially randomized gene library. The ability to synthesize readily such degenerate oligonucleotide sequences according to the prescribed distribution is key to constructing a biased peptide library genuinely reflective of the computational design. Herein we illustrate how a standard DNA synthesizer can be used with only a slight modification to the synthesis protocol to generate a pool of degenerate DNA sequences, which encodes a predetermined amino acid distribution with high fidelity. AbstractProteins exhibit a wide range of physical and chemical properties, including highly selective molecular recognition and catalysis, and are also key components in biological metabolic, catabolic, and signaling pathways. Given that proteins are well-structured and can now be rapidly synthesized, they are excellent targets for engineering of both molecular structure and biological function. Computational analysis of the protein design problem allows scientists to explore sequence space and systematically discover novel protein molecules. Nonetheless, the complexity of proteins, the subtlety of the determinants of folding, and the exponentially large number of possible se...

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Solution structure and dynamics of a de novo designed three-helix bundle protein

Cited by 200 publications

References 59 publications

Getting specificity from simplicity in putative proteins from the prebiotic Earth

Getting specificity from simplicity in putative proteins from the prebiotic Earth

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

Progress in the development and application of computational methods for probabilistic protein design

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