Design of a heme-binding four-helix bundle

Choma, Christin T.; Lear, James D.; Nelson, Mark J.; Dutton, P. Leslie; Robertson, Dan E.; DeGrado, William F.

doi:10.1021/ja00082a005

Cited by 260 publications

(218 citation statements)

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“…18. A 66-residue, amino-terminally cystine-linked coiled coil derived from the leucine zipper region of the protein GCN4 was synthesized and purified by analogy to Choma et al (19). Staphylococcal nuclease (Snase), ␣ subunit of tryptophan synthase (␣-TS), and outer surface protein A (OspA) were gifts of D. Shortle (The Johns Hopkins University, Baltimore), O. Bilsel (University of Massachusetts Medical School, Worcester), C. R. Matthews (University of Massachusetts Medical School), and S. Koide (University of Chicago, Chicago) (6,20).…”

Section: Methodsmentioning

confidence: 99%

Random-coil behavior and the dimensions of chemically unfolded proteins

Kohn

Millett

Jacob

et al. 2004

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

652

751

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Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (RG) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The RG of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble

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Section: Methodsmentioning

confidence: 99%

Random-coil behavior and the dimensions of chemically unfolded proteins

Kohn

Millett

Jacob

et al. 2004

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

652

751

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“…The 48-residue DF1 protein was prepared in good yield by standard solid phase methods used previously for the synthesis of similarly sized designed proteins (37). DF1 adopts a folded helical conformation in aqueous solution, irrespective of its ligation state.…”

Section: Resultsmentioning

confidence: 99%

“…The structures of some simple porphyrin peptide complexes have been solved by NMR (5,41). However, the structures of larger designed proteins with bound cofactors have not been solved, possibly because they have dynamically averaging structures (37,42). Therefore, methods for the design of uniquely folded cofactor-binding proteins are needed.…”

mentioning

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.

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232

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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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“…The density of the proteins in the films (10) decreased substantially when the films were incubated in a buffer with ionic strength higher than 100mM.…”

Section: Discussionmentioning

confidence: 99%

De Novo Design of a CytochromebMaquette for Electron Transfer and Coupled Reactions on Electrodes

et al. 2001

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Experimental explorations of functional mechanisms in natural electron-transfer proteins are often frustrated by their fragility and extreme complexity. We have designed and synthesized four-R-helix-bundle redox proteins, maquettes, that are much simplified and more robust than natural redox proteins and can be designed to bind onto electrode surfaces to facilitate systematic investigations. The points of interest that can be now assessed are not only the processes that govern biological assembly of equilibrium structures, electrochemistry, and electron tunneling rates but also how these factors are coupled together to effect redox driven catalysis. Here we describe maquettes that bis-histidine ligate protoporphyrin IX (heme), much like native b cytochromes, as well as contain charged surface patches, much like native cytochrome c. The positively charged residues aid adsorption to negatively charged surfaces, such as gold electrodes modified by 11-mercaptoundecanoic acid, and facilitate cyclic voltammetry (CV) measurements. CV demonstrates the reversible electrochemistry typical for cytochrome b as well as the coupling of the b-heme oxidation and reduction to proton exchange. The pH dependency of redox midpoint potentials reveals a major (three pH units) shift of the pK a which matches the shift previously shown to originate in nearby glutamates 1 . The redox potentials correspondingly shift from -0.24 (pH > pK red , deprotonated) to -0.11 V (pH < pK ox , protonated). The rate of electron transfer at zero driving force between the hemes and the gold electrode was determined to be 120 s -1 , a rate consistent with tunneling through the mercaptoundecanoic acid spacer and suggesting that the coupled proton exchange is not rate limiting. Reduction of the heme in the presence of CO-saturated buffer shifted the oxidation peak from -0.2 to +0.35 V, indicating massive preferential CO binding to the reduced heme. Consistent with solution spectroscopy, CO must displace one axial histidine to the heme to form the His-CO form of the ferrous heme. The CO is released upon heme oxidation at high potentials. In contrast to coupled proton exchange, CO binding/release and ligand exchange are slow on the time scale of electron tunneling between the heme edge and the electrode.

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Design of a heme-binding four-helix bundle

Cited by 260 publications

References 0 publications

Random-coil behavior and the dimensions of chemically unfolded proteins

Random-coil behavior and the dimensions of chemically unfolded proteins

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

De Novo Design of a CytochromebMaquette for Electron Transfer and Coupled Reactions on Electrodes

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