Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

Development of epistatic YES and AND protein logic gates and their assembly into signalling cascades.

The construction and assembly of artificial allosteric protein switches into information and energy processing networks connected to both biological and non-biological systems is a central goal of synthetic biology and bionanotechnology. However, designing protein switches with the desired input, output and performance parameters is challenging. Here we use a range of reporter proteins to demonstrate that their chimeras with duplicated receptor domains produce YES gate protein switches with large (up to 9,000-fold) dynamic ranges and fast (minutes) response rates. In such switches, the epistatic interactions between largely independent synthetic allosteric sites result in an OFF state with minimal background noise. We used YES gate protein switches based on ß-lactamase to develop quantitative biosensors of therapeutic drugs and protein biomarkers. Furthermore, we demonstrated the reconfiguration of YES gate switches into AND gate switches controlled by two different inputs, and their assembly into signalling networks regulated at multiple nodes.

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices.

Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

Leishmania tarentolae cell-free based approach for rapid anitbody-antigen interaction analysis.

Rapid techniques for producing high-quality recombinant proteins are essential for fast protein functional analysis, as well as various screening applications. Cell-free protein expression is an enabling tool in protein research capable of producing high-quality proteins within a few hours. In this chapter, we describe the use of a Leishmania tarentolae-based cell-free expression system to produce antibody fragments coupled to the analysis of their interaction with their ligands. Interaction analysis is performed using the scalable and sensitive AlphaLISA bead proximity assay. The method presented in this chapter offers a rapid and inexpensive approach for production of putative interacting protein pairs, as well as a multiplexable approach for their rapid interaction analysis.

AbPredict 2: a server for accurate and unstrained structure prediction of antibody variable domains.

SUMMARY: Methods for antibody structure prediction rely on sequence homology to experimentally determined structures. Resulting models may be accurate but are often stereochemically strained, limiting their usefulness in modeling and design workflows. We present the AbPredict 2 web-server, which instead of using sequence homology, conducts a Monte Carlo-based search for low-energy combinations of backbone conformations to yield accurate and unstrained antibody structures. AVAILABILITY AND IMPLEMENTATION: We introduce several important improvements over the previous AbPredict implementation: (i) backbones and sidechains are now modeled using ideal bond lengths and angles, substantially reducing stereochemical strain, (ii) sampling of the rigid-body orientation at the light-heavy chain interface is improved, increasing model accuracy and (iii) runtime is reduced 20-fold without compromising accuracy, enabling the implementation of AbPredict 2 as a fully automated web-server (http://abpredict.weizmann.ac.il). Accurate and unstrained antibody model structures may in some cases obviate the need for experimental structures in antibody optimization workflows.

Prevocational exposure to public health in the Kimberley: a pathway to rural, remote and public health practice.

OBJECTIVE: To evaluate the Kimberley Population Health Unit (KPHU) prevocational public health placement in terms of its contribution to resident medical officers' (RMOs') knowledge, skills, career path and aspirations. DESIGN: All RMOs who had completed a public health placement at the KPHU (n=27) during 2001-2012 were invited to complete an online survey in September 2012. SETTING: The KPHU, based in Broome, provides population health services to the Kimberley region, far north Western Australia. MAIN OUTCOME MEASURES: The extent to which RMOs perceived the development of public health skills and knowledge during the placement, and the degree to which RMOs believe this placement influenced future career pathways and their current practice. RESULTS: Twenty-three RMOs (85%) completed the survey. Sixty per cent are currently working in general practice or public health medicine; of these, 43% have returned to the Kimberley. Over 70% reported that the placement developed their knowledge of public health and Aboriginal health to a 'great' or 'very great' extent. Sixty-one per cent felt that their placement influenced their future desire to work in public health 'a lot' or 'a great extent'. CONCLUSION: This placement provides a unique opportunity for RMOs to undertake public health and Aboriginal health work in a remote setting. Given the increasing demand for prevocational placements, the value of imparting sound public health knowledge to the next generation of doctors and the urgent need to recruit and retain rural doctors, this placement provides a potential model that could be expanded to other locations.

Global health training and postgraduate medical education in Australia: the case for greater integration.

Global health (GH) training is well established overseas (particularly in North America) and reflects an increasing focus on social accountability in medical education. Despite significant interest among trainees, GH is poorly integrated with specialty training programs in Australia. While there are numerous benefits from international rotations in resource-poor settings, there are also risks to the host community, trainee and training provider. Safe and effective placements rely on firm ethical foundations as well as strong and durable partnerships between Australian and overseas health services, educational institutions and GH agencies. More formal systems of GH training in Australia have the potential to produce fellows with the skills and knowledge necessary to engage in regional health challenges in a global context.