How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To evaluate the potential biotechnical value of these proteins, the analysis of protein structures and functions is a central challenge in this program. The stability of protein sample is essential to provide meaningful assay results and increase the crystallizability of the targets. The thermal shift assay (TSA), a fluorescence-based technique, is established as a popular method for optimizing the conditions for protein stability in high-throughput. In TSAs, the employed fluorophores are extrinsic, environmentally-sensitive dyes. An alternative, similar technique is nano differential scanning fluorimetry (nanoDSF), which relies on protein native fluorescence. We present here a novel osmolyte screen, a 96-condition screen of organic additives designed to guide crystallization trials through preliminary TSA experiments. Together with previously-developed pH and salt screens, the set of three screens provides a comprehensive analysis of protein stability in a wide range of buffer systems and additives. The utility of the screens is demonstrated in the TSA and nanoDSF analysis of lysozyme and Protein X, a target protein of the Virus-X project

Multiple roles for plant glutathione transferases in xenobiotic detoxification

Discovered 40 years ago, plant glutathione transferases (GSTs) now have a well-established role in determining herbicide metabolism and selectivity in crops and weeds. Within the GST superfamily, the numerous and plant-specific phi (F) and tau (U) classes are largely responsible for catalyzing glutathione-dependent reactions with xenobiotics, notably conjugation leading to detoxification and, more rarely, bioactivating isomerizations. In total, the crystal structures of 10 plant GSTs have been solved and a highly conserved N-terminal glutathione binding domain and structurally diverse C-terminal hydrophobic domain identified, along with key coordinating residues. Unlike drug-detoxifying mammalian GSTs, plant enzymes utlilize a catalytic serine in place of a tyrosine residue. Both GSTFs and GSTUs undergo changes in structure during catalysis indicative of an induced fit mechanism on substrate binding, with an understanding of plant GST structure/function allowing these proteins to be engineered for novel functions in detoxification and ligand recognition. Several major crops produce alternative thiols, with GSTUs shown to use homoglutathione in preference to glutathione, in herbicide detoxification reactions in soybeans. Similarly, hydroxymethylglutathione is used, in addition to glutathione in detoxifying the herbicide fenoxaprop in wheat. Following GST action, plants are able to rapidly process glutathione conjugates by at least two distinct pathways, with the available evidence suggesting these function in an organ- and species-specific manner. Roles for GSTs in endogenous metabolism are less well defined, with the enzymes linked to a diverse range of functions, including signaling, counteracting oxidative stress, and detoxifying and transporting secondary metabolites

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix

Background: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aqua- 21 porin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is 22 myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic 23 analyses suggested that the sequences 434-452 were a-helical and amphipathic. Methods and Re- 24 sults: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an 25 intrinsically disordered to a more a-helical conformation for the residues 434-467. Recombinantly 26 produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as deter- 27 mined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non- 28 lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to 29 cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compart- 30 ments such as the nuclear and mitochondrial membranes were negative. The N-terminal myristoy- 31 lation of the amphipathic helix appeared not to change either the lipid affinity or membrane locali- 32 sation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it 33 did appear to enhance its helical content. Conclusions: These data support the conclusion that C- 34 terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane 35 binding properties via an adjacent amphipathic helix

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BACKGROUND: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. METHODS AND RESULTS: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. CONCLUSIONS: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix

An amphipathic helix facilitates the membrane binding properties of BFSP1 and its caspase-generated C-terminal domain

Background: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aqua- 21 porin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is 22 myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic 23 analyses suggested that the sequences 434-452 were a-helical and amphipathic. Methods and Re- 24 sults: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an 25 intrinsically disordered to a more a-helical conformation for the residues 434-467. Recombinantly 26 produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as deter- 27 mined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non- 28 lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to 29 cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compart- 30 ments such as the nuclear and mitochondrial membranes were negative. The N-terminal myristoy- 31 lation of the amphipathic helix appeared not to change either the lipid affinity or membrane locali- 32 sation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it 33 did appear to enhance its helical content. Conclusions: These data support the conclusion that C- 34 terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane 35 binding properties via an adjacent amphipathic helix

Derisking the Polymorph Landscape: The Complex Polymorphism of Mexiletine Hydrochloride

This work presents an updated solid form discovery approach to the polymorphism of the anti-arrhythmic drug mexiletine hydrochloride, in which experimental and computational techniques are combined to provide a rigorous characterisation of the solid-form landscape of this compound. The resulting solid forms were characterised by powder and single crystal X-ray diffraction, IR spectroscopy, DSC, and 13C solid-state NMR. This approach reveals five solid form types of mexiletine hydrochloride. Forms 1, 2 and 3 are mutually enantiotropically related anhydrous polymorphs, with Form 1 the room temperature stable form, Form 2 the high temperature form and Form 3 is the thermodynamically stable polymorph between 148 °C and 167 °C. The final two forms termed Types A and B comprise two large families of isomorphous channel solvates, including a fourth non-solvated form isostructural to the type A solvates. We report eleven modifications of each solvate, in which a diverse range of solvents are included in the channels, without changing the fundamental structure of the drug framework. These experimental results go hand-in-hand with computational crystal structure prediction (using the AstraZeneca crystal structure prediction approach), which together suggest that it is unlikely further non-solvated forms, at least with Z' = 1, will be discovered under ambient conditions