Filarete: The Architect of the Renaissance

The transfer of a methyl group is an essential step in many reactions occurring in every living cell. This transformation is catalyzed by methyltransferases (MTases). All MTases share the need for a cofactor to perform their action. S-adenosyl-L-methionine (SAM, AdoMet) is the most frequently used methyl donor in biological systems. Recent studies showed the ability of DNA-C-MTases as well as small molecule-C-MTases to accept SAM analogues for transfer of extended carbon chains to various substrates. Anyway, the improvement towards a broader range of cofactor analogues is of great interest in terms of both biocatalytic synthesis and DNA diagnostics. This proposal aims to combine directed enzyme evolution with rational protein engineering to obtain C-MTases for improved transfer of extended groups from SAM analogues. In general, it is very difficult to develop selection strategies for MTases. The first part of the proposal describes the directed evolution of the DNA-MTase M.SssI towards cofactor analogues by in vitro selection of positive clones. For this purpose the protection of DNA from fragmentation by cognate restriction endonucleases will be used after in vitro expression of gene variants in water- in-oil emulsion droplets (in vitro compartmentalization). This method links genotype and phenotype and provides an effective selection of evolved variants. The result of M.SssI evolution will afterwards be transferred to rational protein engineering of the small molecule MTase NovO. The fact that both enzymes share high structural similarities of their core fold, which include the cofactor binding site, allows the combination of error-prone mutagenesis for directed evolution of M.SssI and site-directed mutagenesis for rational protein design of NovO. Besides analysis of enzyme activities, also biophysical properties of both enzymes will clarify the mechanism of protein-ligand interaction. Therefore dissociation constants of MTases and cofactors will clarify the importance of individual mutations for cofactor specificity. In sum, the proposed work will give new important insights and stimulate prospective applications of alkyl transfer reactions.

Filaretes Libro architettonico, written in Milan around 1460, is the second treatise on architecture written during the Renaissance, following Leon Battista Albertis De re aedificatoria, written around 1450. However, it differs in form and content both from its renowned predecessor and also from the subsequent tradition of architectural treatises in that it is not only written in Italian and richly illustrated, but also uses the Socratic method of dialogue to describe and promote the ideal planning, founding, and construction of a city. Comparison with his more famous predecessor Alberti has in particular led to a neglect of Filarete and his work, with even entire passages of his Libro receiving no attention at all. The task in the first phase of the project consisted of searching for the first time systematically in the holdings of the archives initially in Florence, and then in Milan and Paris for documents relevant to Filarete. The results make it possible to trace, in a partly new and partly more detailed way than previously, Filaretes social position at the court of Francesco Sforza and his path from a largely unsuccessful career as a sculptor and architect to become a literary propagandist for the Renaissance and the eras architect with new insights into the sociology of artists in the mid-fifteenth century in general, a period for which the category of the court artist that has previously been used (Martin Warnke) needs to be corrected. The second phase of the project focused on the text of the Libro architettonico itself, which was subjected for the first time to a detailed and comprehensive iconographic examination. Through a precise reading of previously little-noted or overlooked passages in the context of the text as a whole, on the one hand, and a truly interdisciplinary study of source texts going well beyond the usual art- historical reference points on the other including astronomical/astrological textbooks, the hermetic tradition, manuals of magic, travel literature, and other genres Filaretes Libro architettonico proves to be an outstanding document that crystallizes its time, in an intermediate position between the cultures of Milan and Florence and in the transition between the Middle Ages and the early modern period.