Symmetryâ Directed Selfâ Assembly of a Tetrahedral Protein Cage Mediated by de Novoâ Designed Coiled Coils

The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of proteinâ protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiledâ coil domains to assemble proteins into higherâ order structures. Here, we demonstrate the assembly of a trimeric enzyme into a wellâ defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiledâ coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiledâ coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.Geometry lesson: A modular approach for assembling proteins into largeâ scale geometric structures was developed in which coiledâ coil domains acted as â twist tiesâ to facilitate assembly. The geometry of the cage was specified primarily by the rotational symmetries of the coiled coil and building block protein and was largely independent of protein structural details.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/1/cbic201700406_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/2/cbic201700406.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/3/cbic201700406-sup-0001-misc_information.pd

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane

Front Cover: Symmetryâ Directed Selfâ Assembly of a Tetrahedral Protein Cage Mediated by de Novoâ Designed Coiled Coils (ChemBioChem 19/2017)

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138873/1/cbic201700481.pd

Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant–plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C20-precursor (E,E)-geranyllinalool to the insect-induced C16-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)-geranyllinalool and its C15-analog (E)-nerolidol, which is converted to the respective C11-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase