Keywords Parole chiave: Covalent Linkage, Cu(i) Catalyzed Azide-Alkyne Cycloaddition, De Novo Design, Diiron-Oxo Proteins, Four-Helix Bundles, Heterodimers, Metalloprotein Models,
Affiliations Affiliazioni: *** IBB - CNR ***
University of Napoli Federico II, Napoli, Italy., University of Napoli Federico II, Napoli, Italy; Institute of Biostructures and Bioimages-IBB, CNR, Napoli, Italy., University of Napoli Federico II, Napoli, Italy. Electronic address: firstname.lastname@example.org.,
References Riferimenti: Not available. Non disponibili.
De 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 four-helix bundles through the covalent heterodimerization of two different alpha-helical harpins. In particular, starting from the homodimeric DF3 structure, we developed a protocol for covalently linking the two alpha2 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.