Developing metalloprotein models for diagnostic and sensing applications (original) (raw)
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Proceedings of the National Academy of Sciences, 2013
Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn II < Fe II < Ni II < Co II < Cu II > Zn II ). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn II and Fe II in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe II over Mn II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a twostep process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.
The challenge of metalloproteins: some applications
Suggested running title: The challenge of metalloproteins † ICIQ ‡ IMIM/UPF § Universitat Autònoma de Barcelona 1 ABSTRACT The computational study of metalloproteins faces specific challenges, but holds the promise of important rewards in the form of understanding original enzymatic behaviours which cannot be approached by the usual experimental procedures. The strenghts and limitations of the usual computational approach of studying the reactivity of the metalloprotein with QM or QM/MM studies of the active center of the metalloprotein are discussed with a detailed description of available results on two particular examples: heme oxygen transporters and B 12 cofactors. 2
Journal of Biological Inorganic Chemistry, 1997
The importance of electrostatic effects in determining the free energy of redox reactions in proteins such as cytochromes and iron-sulfur complexes is well established. Several theoretical techniques have been used to analyze how the protein and its environment combine to produce the observed electrochemical midpoints. The free energy of changing the cofactor charge is influenced by the distribution of charges and dipoles in the protein, solvent and ions surrounding the protein, and by the redistribution of these charges and dipoles coupled to the reaction. An outline of a consistent view for calculating these effects will be presented and compared with other theoretical models. Heme redox potentials in yeast cytochrome c and the cytochrome subunit of photosynthetic reaction centers will be calculated to show how these protein structures produce the observed electrochemistry.
Environmental Science & Technology, 2008
Phage-display technology was used to evolve peptides that selectively bind to the metal-oxide hematite (Fe 2 O 3 ) from a library of approximately 3 billion different polypeptides. The sequences of these peptides contained the highly conserved amino acid motif, Ser/Thr-hydrophobic/aromatic-Ser/Thr-Pro-Ser/Thr. To better understand the nature of the peptide-metal oxide binding demonstrated by these experiments, molecular dynamics simulations were carried out for Ser-Pro-Ser at a hematite surface. These simulations show that hydrogen bonding occurs between the two serine amino acids and the hydroxylated hematite surface and that the presence of proline between the hydroxide residues restricts the peptide flexibility, thereby inducing a structural-binding motif. A search of published sequence data revealed that the binding motif (Ser/Thr-Pro-Ser/Thr) is adjacent to the terminal heme-binding domain of both OmcA and MtrC, which are outer membrane cytochromes from the metalreducing bacterium Shewanella oneidensis MR-1. The entire five amino acid consensus sequence (Ser/Thr-hydrophobic/ aromatic-Ser/Thr-Pro-Ser/Thr) was also found as multiple copies in the primary sequences of metal-oxide binding proteins Sil1 and Sil2 from Thalassiosira pseudonana. We suggest that this motif constitutes a natural metal-oxide binding archetype that could be exploited in enzyme-based biofuel cell design and approaches to synthesize tailored metal-oxide nanostructures.
Diiron-containing metalloproteins: Developing functional models
2007
A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technology, such as in degradation of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochemical functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biology, with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-containing proteins, and we developed models for heme proteins and diironeoxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins. To cite this article: O. Maglio et al., C. R. Chimie 10 (2007). Ó 2007 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Transition Metal Induced Conformational Change of Heme Proteins
Spectroscopy Letters, 2014
Binding interaction of transition metals copper, cadmium, and mercury with heme proteins were explored using isothermal calorimetry. Consequent conformational changes of the native proteins analyzed by circular dichroic spectroscopy revealed differential structural alteration of heme proteins. But, significant distortion in myoglobin structure was noted due to bioconjugation with mercury whose greater ionic radius may be responsible for this preferential behavior.
Characterization of interactions and metal ion binding sites in proteins
Current Opinion in Structural Biology, 1994
Recent investigations show that as a class of interactions for designing proteins, hydrophobic interactions are not specific enough, hydrophilic interactions are typically too weak, and water interactions are always on the exterior. In terms of overall protein stability, there is a substantial advantage to a nucleus with strong, directional interactions. Metal ion sites in proteins exhibit strong directional preferences for their coordinate ligands, and the specificities manifested by ions have been demonstrated to be useful in reducing molecular fluctuations. The engineered introduction of zinc binding sites has been shown to improve the stabilities of designed proteins. Metal binding sites can therefore provide important structural building blocks for protein design.
Analyses and Applications of Metalloprotein Complexes
The structural characteristics associated with the binding of beneficial metals (i.e.-Mg 2+ , Zn 2+ and Ca 2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g.-Pb 2+ , Hg 2+ , Cd 2+ , La 3+ , etc.). In this thesis, a statistical analysis of Pb 2+-binding in crystallized protein structures indicates that Pb 2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca 2+ and Pb 2+ indicates that Pb 2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca 2+. The Pb 2+ ion also appears to displace Ca 2+ with little conformational stress in calcium binding proteins (CaBP's). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb 2+ and Gd 3+ will occupy grafted calcium-binding sites with greater affinity than Ca 2+ , and strong evidence is presented to support the hypothesis that Pb 2+ and Gd 3+ will bind non-Working with a cohesive group of talented people always raises the bar of excellence, and I have been fortunate to work with such a group in Dr. Yang's lab. In particular, I wish to thank Ning Chen who gave me an intensive crash course in protein expression and purification, and Dr. Jin Zou for his help in designing methods for analyses of metal binding.
Design of metal cofactors activated by a protein-protein electron transfer system
Proceedings of the National Academy of Sciences of the United States of America, 2006
Protein-to-protein electron transfer (ET) is a critical process in biological chemistry for which fundamental understanding is expected to provide a wealth of applications in biotechnology. Investigations of protein-protein ET systems in reductive activation of artificial cofactors introduced into proteins remains particularly challenging because of the complexity of interactions between the cofactor and the system contributing to ET. In this work, we construct an artificial protein-protein ET system, using heme oxygenase (HO), which is known to catalyze the conversion of heme to biliverdin. HO uses electrons provided from NADPH/cytochrome P450 reductase (CPR) through protein-protein complex formation during the enzymatic reaction. We report that a Fe(III)(Schiff-base), in the place of the active-site heme prosthetic group of HO, can be reduced by NADPH/CPR. The crystal structure of the Fe(10-CH(2)CH(2)COOH-Schiff-base).HO composite indicates the presence of a hydrogen bond between ...