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Vincenzo Pavone: Friend, mentor and inspiring scientist
No full textEditorial for this special issue of Biopolymers dedicated to Prof. Vincenzo Pavone, in celebration of his 65th birthday. The reviews and original articles included in this issue are contributed by friends, collaborators and former students of Vincenzo. They pay tribute to his outstanding career, research accomplishments, and valuable role as mentor and inspiring scientist
Designing Covalently Linked Heterodimeric Four-Helix Bundles
No full textDe novo design has proven a powerful methodology for understanding protein folding
and function, and for mimicking or even bettering the properties of natural proteins.
Extensive progress has been made in the design of helical bundles, simple structural
motifs that can be nowadays designed with a high degree of precision. Among helical
bundles, the four-helix bundle is widespread in nature, and is involved in numerous and
fundamental processes. Representative examples are the carboxylate bridged diiron
proteins, which perform a variety of different functions, ranging from reversible dioxygen
binding to catalysis of dioxygen-dependent reactions, including epoxidation desaturation, monohydroxylation, and radical formation. The “Due Ferri” (two-irons; DF) family of proteins is the result of a de novo design approach, aimed to reproduce in
minimal four-helix bundle models the properties of the more complex natural diiron
proteins, and to address how the amino acid sequence modulates their functions.
The results so far obtained point out that asymmetric metal environments are essential
to reprogram functions, and to achieve the specificity and selectivity of the natural
enzymes. Here, we describe a design method that allows constructing asymmetric fourhelix
bundles through the covalent heterodimerization of two different α-helical
harpins. In particular, starting from the homodimeric DF3 structure, we developed a
protocol for covalently linking the two α2 monomers by using the Cu(I) catalyzed
azide–alkyne cycloaddition. The protocol was then generalized, in order to include the
construction of several linkers, in different protein positions. Our method is fast, low cost,
and in principle can be applied to any couple of peptides/proteins we desire to link
Peptides and metal ions: A successful marriage for developing artificial metalloproteins
Full text linkThe mutual relationship between peptides and metal ions enables metalloproteins to have crucial roles in biological systems, including structural, sensing, electron transport, and catalytic functions. The effort to reproduce or/and enhance these roles, or even to create unprecedented functions, is the focus of protein design, the first step toward the comprehension of the complex machinery of nature. Nowadays, protein design allows the building of sophisticated scaffolds, with novel functions and exceptional stability. Recent progress in metalloprotein design has led to the building of peptides/proteins capable of orchestrating the desired functions of different metal cofactors. The structural diversity of peptides allows proper selection of first- and second-shell ligands, as well as long-range electrostatic and hydrophobic interactions, which represent precious tools for tuning metal properties. The scope of this review is to discuss the construction of metal sites in de novo designed and miniaturized scaffolds. Selected examples of mono-, di-, and multi-nuclear binding sites, from the last 20 years will be described in an effort to highlight key artificial models of catalytic or electron-transfer metalloproteins. The authors' goal is to make readers feel like guests at the marriage between peptides and metal ions while offering sources of inspiration for future architects of innovative, artificial metalloproteins
Structural and functional aspects of metal binding sites in natural and designed metalloproteins
No full textThis book chapter discusses the main properties of metal ions in proteins. First, it describes the amino acids that act as ligands and their possible binding modes. The most representative metal ions in biological systems are briefly
outlined, mainly regarding their preferred geometry and their functions.
Finally, the chapter focuses on two classes of iron-containing metalloproteins (heme proteins and carboxylate-bridged diiron proteins) in order to illustrate how the same metal cofactor can be engaged in a number of different roles. The various first and second ligation sphere interactions, which finely tune the cofactor properties, thus effecting such different functions, will be highlighted.
The models developed by us for these two classes of metalloproteins will be also described, summarizing principles and methods for designing artificial metalloproteins
De Novo Design of Four-Helix Bundle Metalloproteins: One Scaffold, Diverse Reactivities
Full text linkDe novo protein design represents an attractive approach for testing and extending our understanding of metalloprotein structure and function. Here, we describe our work on the design of DF (Due Ferri or two-iron in Italian), a minimalist model for the active sites of much larger and more complex natural diiron and dimanganese proteins. In nature, diiron and dimanganese proteins protypically bind their ions in 4-Glu, 2-His environments, and they catalyze diverse reactions, ranging from hydrolysis, to O2-dependent chemistry, to decarbonylation of aldehydes. In the design of DF, the position of each atom-including the backbone, the first-shell ligands, the second-shell hydrogen-bonded groups, and the well-packed hydrophobic core-was bespoke using precise mathematical equations and chemical principles. The first member of the DF family was designed to be of minimal size and complexity and yet to display the quintessential elements required for binding the dimetal cofactor. After thoroughly characterizing its structural, dynamic, spectroscopic, and functional properties, we added additional complexity in a rational stepwise manner to achieve increasingly sophisticated catalytic functions, ultimately demonstrating substrate-gated four-electron reduction of O2 to water. We also briefly describe the extension of these studies to the design of proteins that bind nonbiological metal cofactors (a synthetic porphyrin and a tetranuclear cluster), and a Zn2+/proton antiporting membrane protein. Together these studies demonstrate a successful and generally applicable strategy for de novo metalloprotein design, which might indeed mimic the process by which primordial metalloproteins evolved. We began the design process with a highly symmetrical backbone and binding site, by using point-group symmetry to assemble the secondary structures that position the amino acid side chains required for binding. The resulting models provided a rough starting point and initial parameters for the subsequent precise design of the final protein using modern methods of computational protein design. Unless the desired site is itself symmetrical, this process requires reduction of the symmetry or lifting it altogether. Nevertheless, the initial symmetrical structure can be helpful to restrain the search space during assembly of the backbone. Finally, the methods described here should be generally applicable to the design of highly stable and robust catalysts and sensors. There is considerable potential in combining the efficiency and knowledge base associated with homogeneous metal catalysis with the programmability, biocompatibility, and versatility of proteins. While the work reported here focuses on testing and learning the principles of natural metalloproteins by designing and studying proteins one at a time, there is also considerable potential for using designed proteins that incorporate both biological and nonbiological metal ion cofactors for the evolution of novel catalysts
Miniaturized metalloproteins: a designed rubredoxin-like peptide housing a reversible Fe(II)/Fe(III) couple
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