Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.DOI: http://dx.doi.org/10.7554/eLife.12125.001

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A novel concept for a biofuel cell is presented. Enzyme based fuel cells suffer from enzyme instability when a long time of operation is required. Hence, a system that will continuously produce the biocatalyst needed for the system is necessary. A hybrid of an enzyme-based microbial fuel cell was developed. The redox enzyme glucose oxidase from Aspergillus niger was displayed on the surface of Saccharomyces cerevisiae using the Yeast Surface Display System in a high copy number and as an active enzyme. We have demonstrated its activity both biochemically and electrochemically and observed much higher activity over yeast cells not displaying glucose oxidase as well as over purified glucose oxidase from Aspergillus niger. Further, we were able to construct a biofuel cell, where the anode was comprised of the yeast cells displaying glucose oxidase in the presence of a mediator (methylene blue) and the cathode compartment was comprised of the oxygen reducing enzyme laccase from Trametes versicolor and a redox mediator. Our constructed biofuel cell displayed higher power outputs and current densities than those observed for unmodified yeast and a much longer time of operation in comparison with a similar cell where the anode is comprised of purified glucose oxidase.

Dynamic switching of PCNA-partner interactions is essential for normal DNA replication and repair in yeast.

The structure and connectivity of protein-protein interaction (PPI) networks are maintained throughout evolution by coordinated changes (coevolution) of network proteins. Despite extensive research, relatively little is known regarding the molecular basis and functional implications of the coevolution of PPI networks. Here, we used proliferating cell nuclear antigen, a hub protein that mediates DNA replication and repair in eukaryotes, as a model system to study the coevolution of PPI networks in fungi. Using a combined bioinformatics and experimental approach, we discovered that PCNA-partner interactions tightly coevolved in fungal species, leading to specific modes of recognition. We found that fungal proliferating cell nuclear antigen-partner interaction networks diverged into two distinct groups as a result of such coevolution and that hybrid networks of these groups are functionally noncompatible in Saccharomyces cerevisiae. Our results indicate that the coevolution of PPI networks can form functional barriers between fungal species, and thus can promote and fix speciation.interdomain connecting loop | yeast two hybrid | directed evolution P rotein-protein interaction (PPI) networks play vital roles in executing almost all essential biological processes. The availability of sequencing data, as well as high-throughput experimental approaches to identify and generate comprehensive maps of PPIs, has enabled the development of a network-based view of biological processes (1, 2). Such networks are composed of ensembles of proteins that act together in a coordinated manner to execute a variety of essential biological processes, such as DNA replication, transcription, and signal transduction. In many cases, such networks are modular and contain highly connected proteins, termed "hub proteins," that can regulate a given biological process by switching partners with high spatial and temporal resolution (3).Many PPI networks are conserved over evolutionary time scales to promote a variety of biological processes in different organisms (4). One mechanism to maintain network structure and connectivity throughout evolution involves the coevolution of interacting proteins through coordinated changes in protein-protein interfaces (5). The coevolution of interacting proteins can form reproductive barriers between organisms as a result of hybrid network incompatibility, and thus can be an important driving force in promoting and fixing speciation. Currently, the study of coevolution of PPI networks is extremely challenging because of difficulties in identifying and characterizing coordinated sequence changes in network proteins during natural evolution. Even if such sequence changes are detected, their functional implications are difficult to predict. Thus, relatively little is known overall regarding the dynamics and functional importance of the coevolution of hubpartner interactions across different species.In eukaryotes, DNA replication and repair processes are mediated by proliferating cell nuclear antigen (PCNA) throug...