optimized-production-analysis-recombinant-protein-filled-vesicles

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from <em>E. coli</em>

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an ...

University of Kent

The timing of cell division is controlled by the coupled regulation of growth and division. The target of rapamycin (TOR) signalling network synchronises these processes with the environmental setting. Here, we describe a novel interaction of the fission yeast TOR complex 2 (TORC2) with the cytokinetic actomyosin ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that the myosin II protein Myp2 and the myosin V protein Myo51 play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2-dependent phosphorylation of actin-capping protein 1 (Acp1, a known regulator of cytokinesis) controls CAR stability, modulates Acp1-Acp2 (the equivalent of the mammalian CAPZA-CAPZB) heterodimer formation and is essential for survival upon stress. Thus, TORC2 localisation to the CAR, and TORC2-dependent Acp1 phosphorylation contributes to timely control and the fidelity of cytokinesis and cell division.

TOR complex 2 localises to the cytokinetic actomyosin ring and controls the fidelity of cytokinesis.

Amino-terminal acetylation is a ubiquitous protein modification affecting the majority of eukaryote proteins to regulate stability and function. We describe an optimised recombinant expression system for rapid production of amino terminal-acetylated proteins within bacteria. We go on to describe the system's use in a fluorescence based in vivo assay for use in the high-throughput screen to identify drugs that impact amino-terminal acetylation-dependent oligomerisation. These new tools and protocols will allow researchers to enhance routine recombinant protein production and identify new molecules for use in research and clinical applications.

An enhanced recombinant amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affecting α-synuclein oligomerisation.

Biofilm-associated proteins (BAPs) are important for early biofilm formation (adhesion) by bacteria and are also found in mature biofilms. BapA from Salmonella is a ~386-kDa surface protein, comprising 27 tandem repeats predicted to be bacterial Ig-like (BIg) domains. Such tandem repeats are conserved for BAPs across different bacterial species, but the function of these domains is not completely understood. In this work, we report the first study of the mechanical stability of the BapA protein. Using magnetic tweezers, we show that the folding of BapA BIg domains requires calcium binding and the folded domains have differential mechanical stabilities. Importantly, we identify that >100 nM concentration of calcium is needed for folding of the BIg domains, and the stability of the folded BIg domains is regulated by calcium over a wide concentration range from sub-micromolar (μM) to millimolar (mM). Only at mM calcium concentrations, as found in the extracellular environment, do the BIg domains have the saturated mechanical stability. BapA has been suggested to be involved in Salmonella invasion, and it is likely a crucial mechanical component of biofilms. Therefore, our results provide new insights into the potential roles of BapA as a structural maintenance component of Salmonella biofilm and also Salmonella invasion.

Calcium-mediated Protein Folding and Stabilization of Salmonella Biofilm-associated Protein A.

Cells respond to changes in their environment through signaling networks that modulate cytoskeleton and membrane organization to coordinate cell-cycle progression, polarized cell growth and multicellular development. Here, we define a novel regulatory mechanism by which the motor activity and function of the fission yeast type one myosin, Myo1, is modulated by TORC2-signalling-dependent phosphorylation. Phosphorylation of the conserved serine at position 742 (S742) within the neck region changes both the conformation of the neck region and the interactions between Myo1 and its associating calmodulin light chains. S742 phosphorylation thereby couples the calcium and TOR signaling networks that are involved in the modulation of myosin-1 dynamics to co-ordinate actin polymerization and membrane reorganization at sites of endocytosis and polarised cell growth in response to environmental and cell-cycle cues.

TORC2-Gad8-dependent myosin phosphorylation modulates regulation by calcium.

Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining filamentous actin (F-actin) and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally diverse compounds. After reducing the dimensionality of these data, only ∼25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2,003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind the focal adhesion protein, talin. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially divergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks, where the theoretical plasticity of interactions is nearly infinite, yet phenotypes in vivo are constrained into a limited subset of practicable configurations.

High-Content Imaging of Unbiased Chemical Perturbations Reveals that the Phenotypic Plasticity of the Actin Cytoskeleton Is Constrained.

Talin and vinculin are part of a multicomponent system involved in mechanosensing in cell-matrix adhesions. Both exist in autoinhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talin's interaction with vinculin has not been investigated. Here by quantifying the kinetics of force-dependent talin-vinculin interactions using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin, resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an autoinhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

Force-Dependent Interactions between Talin and Full-Length Vinculin.

Calmodulin is a conserved calcium signalling protein that regulates a wide range of cellular functions. Amino-terminal acetylation is a ubiquitous post-translational modification that affects the majority of human proteins, to stabilise structure, as well as regulate function and proteolytic degradation. Here, we present data on the impact of amino-terminal acetylation upon structure and calcium signalling function of fission yeast calmodulin. We show that NatA-dependent acetylation stabilises the helical structure of the Schizosaccharomyces pombe calmodulin, impacting its ability to associate with myosin at endocytic foci. We go on to show that this conserved modification impacts both the calcium-binding capacity of yeast and human calmodulins. These findings have significant implications for research undertaken into this highly conserved essential protein.

Acetylation stabilises calmodulin-regulated calcium signalling.

Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5-14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54-1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage.

We describe an innovative system that exports diverse recombinant proteins in membrane-bound vesicles from . These recombinant vesicles compartmentalize proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulfide-bond containing proteins from bacteria. The release of vesicle-packaged proteins supports isolation from the culture and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.

High-yield vesicle-packaged recombinant protein production from .

Antimicrobial resistance is one of the greatest threats to human health. Gram-positive methicillin resistant  (MRSA), in both its planktonic and biofilm form, is of particular concern. Herein we identify the hydrogelation properties for a series of intrinsically fluorescent, structurally related supramolecular self-associating amphiphiles and determine their efficacy against both planktonic and biofilm forms of MRSA. To further explore the potential translation of this hydrogel technology for real-world applications, the toxicity of the amphiphiles was determined against the eukaryotic multicellular model organism, . Due to the intrinsic fluorescent nature of these supramolecular amphiphiles, material characterisation of their molecular self-associating properties included; comparative optical density plate reader assays, rheometry and widefield fluorescence microscopy. This enabled determination of amphiphile structure and hydrogel sol dependence on resultant fibre formation.

Controlling the structure of supramolecular fibre formation for benzothiazole based hydrogels with antimicrobial activity against methicillin resistant .

Karen Baker

School of Biosciences

University of Kent

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

Karen Baker

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University of Kent

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

School of Biosciences