A monomeric StayGold fluorescent protein.

StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric.

Activator-induced conformational changes regulate division-associated peptidoglycan amidases.

AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX-EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX-EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.

Development and proposed evaluation of an eHealth learning tool for undergraduate university students in Ireland.

Undergraduate university students are at a critical stage of development in terms of their academic, social, psychological and behavioural health. Patterns established during these formative years can last a lifetime. eHealth tools have the potential to be engaging, convenient and accessible to a wide range of students by providing health information and enhancing the uptake of positive health behaviours. The 'Healthy Trinity Online Tool' (H-TOT) was developed in collaboration with students and a transdisciplinary team with decades of experience between them in terms of research, clinical responsibility and service delivery. Developmental steps undertaken included: a literature review to formulate the topic content choices; a survey of students to check the relevance and suitability of topics identified; and, the tacit experience of the development team. This co-design model led to the development of content encompassing academic life, healthy eating, physical activity, mood, financial matters, alcohol, tobacco, drugs and relaxation. Qualitative focus groups were subsequently conducted for in-depth exploration of the usage and functionality of H-TOT. The theoretical underpinnings include the locus of control and social cognitive theory. Evidence-based behavioural change techniques are embedded throughout. During early pre-piloting of H-TOT, the team identified and solved content functionality problems. The tone of the content was also revised to ensure it was non-judgemental. To make the H-TOT as interactive as possible, video scenarios were included and all content was audio-recorded to allow playback for students with visual or learning difficulties. Evaluation plans for the pilot year of H-TOT are outlined.

Insights into bacterial cell division from a structure of EnvC bound to the FtsX periplasmic domain.

FtsEX is a bacterial ABC transporter that regulates the activity of periplasmic peptidoglycan amidases via its interaction with the murein hydrolase activator, EnvC. In Escherichia coli, FtsEX is required to separate daughter cells after cell division and for viability in low-osmolarity media. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for amidase activation, but the process itself is poorly understood. Here we present the 2.1 Å structure of the FtsX periplasmic domain in complex with its periplasmic partner, EnvC. The EnvC-FtsX periplasmic domain complex has a 1-to-2 stoichiometry with two distinct FtsX-binding sites located within an antiparallel coiled coil domain of EnvC. Residues involved in amidase activation map to a previously identified groove in the EnvC LytM domain that is here found to be occluded by a "restraining arm" suggesting a self-inhibition mechanism. Mutational analysis, combined with bacterial two-hybrid screens and in vivo functional assays, verifies the FtsEX residues required for EnvC binding and experimentally test a proposed mechanism for amidase activation. We also define a predicted link between FtsEX and integrity of the outer membrane. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for resistance to membrane-attacking antibiotics and detergents to which E. coli would usually be considered intrinsically resistant. These structural and functional data provide compelling mechanistic insight into FtsEX-mediated regulation of EnvC and its downstream control of periplasmic peptidoglycan amidases.