De novodesigned cyclic-peptide heme complexes

Abstract

The structural characterization of de novo designed metalloproteins together with determination of chemical reactivity can provide a detailed understanding of the relationship between protein structure and functional properties. Toward this goal, we have prepared a series of cyclic peptides that bind to water-soluble metalloporphyrins (Fe III and Co III ). Neutral and positively charged histidine-containing peptides bind with a high affinity, whereas anionic peptides bind only weakly to the negatively charged metalloporphyrin. Additionally, it was found that the peptide becomes helical only in the presence of the metalloporphyrin. CD experiments confirm that the metalloporphyrin binds specific cyclic peptides with high affinity and with isodichroic behavior. Thermal unfolding experiments show that the complex has ''native-like'' properties. Finally, NMR spectroscopy produced well dispersed spectra and experimental restraints that provide a high-resolution solution structure of the complexed peptide.O ne approach to understanding the folding and function of proteins is to attempt their design from first principles (1-3). Such design, however, requires not only incorporation of nonpolar interactions, but also inclusion of specific hydrogen bonds, disulfide bridges, ion pairs, and metal chelation. Without these interactions, proteins form compact folding intermediates referred to as molten globules (4). Molten-globule structures are compact and possess a high degree of secondary structure, but they are also highly dynamic, with both internal and external side chains rapidly interconverting among a large family of rotamers. Analysis of protein structures, on the other hand, reveals that the interior hydrophobic core is well packed and rigid. Therefore, two challenges occur in the design of ''native-like'' proteins. By using positive design (3, 5-7), one can take into account issues such as hydrophobicity and the helix-or sheet-forming propensity of amino acids. By using negative design [a term developed to describe the selective introduction of unique stabilizing interactions (2, 3)], one can introduce features that stabilize one specific fold, while, at the same time, they destabilize alternative topologies. By using this dual strategy, one can increase the free-energy gap between the native and molten-globule states.With the challenge of design making rapid progress, several groups have begun to look at the incorporation of functional cofactors in hopes of producing de novo catalytic proteins (8-14). Many enzymes require mono-and dinuclear metal ions (15), metal clusters (ferredoxin and nitrogenase) (16), heme (cytochromes and globins) (17), and organic cofactors (flavin-and quinone-based enzymes) (18). These cofactors are important, especially when processes such as oxidation͞reduction or substrate binding and activation are required (15).Studies of natural enzymes have led to considerable understanding of their structure and function. Reduction of these complicated systems to minimal models, however, will allow ...

In Vitro Studies on Thioether Bond Formation between Hydrogenobacter thermophilus Apocytochrome c552 with Metalloprotoporphyrin Derivatives

“…Thioether bonds confer stability on protein structure (1), and therefore, the ability to attach metalloporphyrins covalently to engineered proteins offers the possibility of achieving catalytic function in a novel heme-derivatized protein. The de novo designed metalloproteins can also provide a detailed understanding of the relationship between protein structure and functional properties (31).…”

Modulation of function in a minimalist heme-binding membrane protein

“…Water-soluble de novo designed heme proteins have long been used as models of more complicated, membraneembedded natural proteins that are involved in energy transduction [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Several factors, including heme solvent exposure [9,10], stabilization of the peptide scaffold [2,6,15], and local electrostatic interactions [7], have been shown to modulate the redox potential and proton coupling of the cofactor in these models.…”

Structural propensities in the heme binding region of apocytochrome b5. II. Heme conjugates

“…The influence of electrostatics on affinity has been documented in the case of cyclic peptides. 38 A similar explanation may hold for the low tendency of BHC-22C 0 to exhibit bis-histidine ligation and refolding. TFE was used to shift the conformational equilibria towards holoprotein-like structure and further discriminate the conjugated and unconjugated peptides.…”