ItemDevelopment of a whole-cell biosensor for β-lactamase inhibitor discovery(The Royal Society of Chemistry, 2023-10-02) Jeffs, Mitchell A.; Gray, Rachel A. V.; Sheth, Prameet; Lohans, Christopher T.The production of β-lactamases by bacterial pathogens endangers antimicrobial therapy, and new inhibitors for β-lactamases are urgently needed. We report the development of a luminescentbased biosensor that quantifies β-lactamase inhibition in a cellular context, based on the activation of transcriptional factor AmpR following the exposure of bacterial cells to β-lactams. This rapid method can account for factors like membrane permeability and can be employed to identify new β-lactamase inhibitors. ItemProtein Engineering of Antifreeze Proteins Reveals that Their Activity Scales with the Area of the Ice-Binding Site(Wiley, 2022-12-02) Scholl, Connor L.; Davies, PeterAntifreeze proteins (AFPs) protect organisms from freezing by binding to ice crystals to prevent their growth. Here, we have investigated how the area of an AFP's ice-binding site (IBS) changes its antifreeze activity. The polyproline type II helical bundle fold of the 9.6-kDa springtail (Collembola) AFP from Granisotoma rainieri (a primitive arthropod) facilitates changes to both IBS length and width. A one quarter decrease in area reduced activity to less than 10%. A one quarter increase in IBS width, through the addition of a single helix, tripled antifreeze activity. However, increasing IBS length by a similar amount actually reduced activity. Expanding the IBS area can greatly increase antifreeze activity but needs to be evaluated by experimentation on a case-by-case basis. ItemHydrogen Peroxide and Phosphoinositide Metabolites Synergistically Regulate a Cation Current to Influence Neuroendocrine Cell Bursting(Wiley, 2021-10-22) Chauhan-Puri, Alamjeet K.; Lee, Kelly H.; Magoski, NeilIn various neurons, including neuroendocrine cells, non-selective cation channels elicit plateau potentials and persistent firing. Reproduction in the marine snail, Aplysia californica, is initiated when the neuroendocrine bag cell neurons undergo an afterdischarge, i.e., a prolonged period of enhanced excitability and spiking during which egg-laying hormone is released into the blood. The afterdischarge is associated with both the production of hydrogen peroxide (H2O2) and activation of phospholipase C (PLC), which hydrolyzes phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3). We previously demonstrated that H2O2 gates a voltage-dependent cation current and evokes spiking in bag cell neurons. The present study tests if DAG and IP3 impact the H2O2-induced current and excitability. In whole-cell voltage-clamped cultured bag cell neurons, bath-application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the H2O2-induced current, which was amplified by the inclusion of IP3 in the pipette. A similar outcome was produced by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. In current-clamp, OAG or OAG plus IP3, elevated the frequency of H2O2-induced bursting. PKC is also triggered during the afterdischarge; when PKC was stimulated with phorbol 12-myristate 13-acetate, it caused a voltage-dependent inward current with a reversal potential similar to the H2O2-induced current. Furthermore, PKC activation followed by H2O2 reduced the onset latency and increased the duration of action potential firing. Finally, inhibiting nicotinamide adenine dinucleotide phosphate oxidase with 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine diminished evoked bursting in isolated bag cell neuron clusters. These results suggest that reactive oxygen species and phosphoinostide metabolites may synergize, and contribute to reproductive behaviour by promoting neuroendocrine cell firing. ItemThese Motors Were Made for Walking(Wiley, 2020-05-29) Hunter, Byron; Allingham, JohnKinesins are a diverse group of ATP‐dependent motor proteins that transport cargos along microtubules (MTs) and change the organization of MT networks. Shared among all kinesins is a ~ 40 kDa motor domain that has evolved an impressive assortment of motility and MT remodeling mechanisms as a result of subtle tweaks and edits within its sequence. Several elegant studies of different kinesin isoforms have exposed the purpose of structural changes in the motor domain as it engages and leaves the MT. However, few studies have compared the sequences and MT contacts of these kinesins systematically. Along with clever strategies to trap kinesin‐tubulin complexes for X‐ray crystallography, new advancements in cryo‐electron microscopy have produced a burst of high‐resolution structures that show kinesin–MT interfaces more precisely than ever. This review considers the MT interactions of kinesin subfamilies that exhibit significant differences in speed, processivity, and MT remodeling activity. We show how their sequence variations relate to their tubulin footprint and, in turn, how this explains the molecular activities of previously characterized mutants. As more high‐resolution structures become available, this type of assessment will quicken the pace toward establishing each kinesin’s design‐function relationship. ItemA Fluorescence‐Based Assay for Screening β‐Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2(Wiley, 2019) de Munnik, Mariska; Lohans, Christopher T.; Langley, Gareth W.; Bon, Corentin; Brem, Jürgen; Schofield, Christopher J.Mycobacterium tuberculosis l,d‐transpeptidases (Ldts), which are involved in cell‐wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence‐based assay for LdtMt2, which is suitable for high‐throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2, thus making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of β‐lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β‐lactams are more potent Ldt inhibitors than other β‐lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure–activity relationship studies on Ldts, thereby facilitating the identification of new antibiotics active against M. tuberculosis.