Multicellular biology, as well as new cell based therapies, are dependent on the control of cell-cell interactions. Herein, we detail the use of a multivalent lipidated scaffold for the rapid...

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast-and mammalian-based systems have demonstrated FTase

Despite broad interest in understanding the biological implications of protein farnesylation in regulating different facets of cell biology, the use of this post-translational modification to develop protein-based materials and therapies remains underexplored. The progress has been slow due to the lack of accessible methodologies to generate farnesylated proteins with broad physicochemical diversities rapidly. This limitation, in turn, has hindered the empirical elucidation of farnesylated proteins’ sequence–structure–function rules. To address this gap, we genetically engineered prokaryotes to develop operationally simple, high-yield biosynthetic routes to produce farnesylated proteins and revealed determinants of their emergent material properties (nano-aggregation and phase-behavior) using scattering, calorimetry, and microscopy. These outcomes foster the development of farnesylated proteins as recombinant therapeutics or biomaterials with molecularly programmable assembly.

Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its trans- and cis-form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in Saccharomyces cerevisiae. One photoswitchable analog of farnesyl diphosphate (AzoFPP-1) allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs in vitro and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility of this approach for the optical control of protein prenylation.

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