vector popine p116  (Addgene inc)

Journal: bioRxiv

Article Title: P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

doi: 10.1101/2023.10.24.563710

Figure Lengend Snippet: a, Mass spectrometry analysis of DPPC extraction from liposomes by P116. Mass spectra of empty P116 (blue; traces of other lipids of mass 713, 738, 743 and 757 Da are visible), DPPC carryover after liposome pelleting (red; no DPPC contamination is visible) and P116 after incubation with and pelleting of DPPC liposomes (black; a clear peak at 734 Da, the mass of DPPC, is visible). The DPPC peak is annotated. Only the final sample of P116 contains DPPC. b, Single-particle cryo-EM structure of the truncated P116, i.e. without the N-terminal domain, has a similar fold to the full-length protein. The cryo-EM density of P116 without the N-terminal domain (246–818) at 4.16 Å resolution superimposed on the ribbon representation of the filled full-length ectodomain of P116 (60–868) (PDB: 8A9A). c, The single-particle cryo-EM structure shows that the truncated P116 subjected to the refilling procedure did not take up lipids and stayed in the empty conformation. Superimposition of the P116 (246–818) dimer in the filled state (dark purple, dark orange) with the P116 (246–818) monomer after emptying and refilling (light purple, light orange). Helix pair 2 of the filled conformation is clearly different from the conformation of the truncated P116 monomer density, illustrating that the monomers were not refilled but remained empty.

Article Snippet: The extracellular domain of P116, C-terminally shortened and HIS-tagged (30– 957), was expressed via the vector pOPINE_P116 backbone #26043; Addgene, Watertown, USA).

Techniques: Mass Spectrometry, Extraction, Liposomes, Incubation, Single Particle, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

doi: 10.1101/2023.10.24.563710

Figure Lengend Snippet: a, Tomographic slice (0.837 Å/pix) of M. pneumoniae cells overexpressing p116 from its native promoter. b, 30 Å average of 700 particles (grey surface, EMD-18629), overlayed with P116 in a conformation obtained from MD simulations. The distance between the N-terminus and the membrane is 100 Å. c, Flexibility of P116 (60–868) simulated close to a membrane. The histograms of range of motion from MD simulations show an even larger flexibility than the range of motion observed in single-particle structures of solvated P116 (indicated by grey bars). Three parameters are displayed: The wringing angle (between monomers), the arc diameter (angle between the monomers) and the angle of the N-terminal to the core domain. The wringing between monomers was dramatically increased in the vicinity of a membrane, allowing the monomers to face almost opposite directions. Down-down and up-down refer to the directions the central clefts of each monomer are facing. The angle between the N-terminal domain and the core domain was fixed at ∼120°–135° (grey bars) in single-particle structures, while it ranged between 0° and 125° in simulated empty P116 in the vicinity of a membrane. Stable membrane docking restricted the flexibility range of the docked monomer. d, Renders from MD simulation of P116 (60–868). The attachment of P116 to a membrane involves phenylalanine residues (yellow beads) in the N-terminal domain. D-docking of monomer 1 precedes N-docking through insertion of F227 and F214 (shown in yellow) of monomer 2. The protein is represented as a filled volume colored by domain. The membrane (DOPG:DOPE:DOPC, 40:30:30) is represented by the phosphate moieties of the lipids, shown as silver beads. Water, ions, and lipid tails are not shown for clarity.

Article Snippet: The extracellular domain of P116, C-terminally shortened and HIS-tagged (30– 957), was expressed via the vector pOPINE_P116 backbone #26043; Addgene, Watertown, USA).

Techniques: Membrane, Single Particle

Journal: bioRxiv

Article Title: P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

doi: 10.1101/2023.10.24.563710

Figure Lengend Snippet: In all simulation renders, P116 is represented by a filled volume or cartoon representation colored by domain. When present, the membrane is indicated by the phosphate moiety (represented by silver beads) and solvated lipids are represented by phosphate moieties (blue beads) and tails (red beads). Water and ions are not shown for clarity. a, D-docking places the tip of the DCA near the membrane. b, N-docking places the gate of the DCA near the membrane when closed (left) and opened (right). Residues F860, F856, F227 and F214, which insert into the membrane, are shown as yellow beads. c, Details of DCA architecture show opening at the gate side only. Comparison of the DCA conformation between the filled and empty single-particle cryo-EM structures (PDB: 8A9A and 8A9B, respectively). The two helices comprising the DCA are shown in blue with the individual amino acid sidechains as stick models. The surface representations are colored by hydrophobicity factor (yellow is hydrophobic and blue is hydrophilic). The DCA opens and closes at the gate side while the tip side remains closed. d, N-docking promotes opening of the membrane below the DCA. Top view of the membrane during double docking of P116 (P116 not shown for clarity). The D-docking area is labelled with a blue box, the N-docking area with a red box. Residues F860, F856, F227 and F214 are shown as yellow beads. The membrane barrier is perturbed only during N-docking, which promotes opening of the membrane below the DCA. e, Lipids enter the DCA in a split position. e 1 Coarse-grained MD simulation of P116 with free-floating DOPE, DOPC and DOPG lipids. F860, F856, F227 and F214 are shown as yellow beads. (Left) Start of simulation with evenly distributed lipids. (Right) DOPE lipid entering the cavity through the DCA. The lipid is shown as a filled volume colored according to the trajectory frame. e 2 (Left) N-docked P116 with DOPE lipid in split position placed below the DCA. (Right) DOPE lipid entering the cavity through the DCA. The taken-up lipid is represented by beads, head group in cyan and tail in magenta for contrast. The DCA and finger helices of the monomer involved in N-docking and uptake are rendered as transparent shapes for clarity. f, Cargo inside the cavity is positioned inside the DCA. Atomistic MD simulation of 12 cholesterol molecules (shown as red spheres) in the cavity of P116. f 1 Start of simulation with cholesterol molecules evenly distributed inside the cavity. f 2 Cholesterol molecules rearrange and are squeezed into the cavity towards the dimerization domain. f 3 An individual cholesterol molecule inserts in the hydrophobic channel formed by the DCA.

Article Snippet: The extracellular domain of P116, C-terminally shortened and HIS-tagged (30– 957), was expressed via the vector pOPINE_P116 backbone #26043; Addgene, Watertown, USA).

Techniques: Membrane, Comparison, Single Particle, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

doi: 10.1101/2023.10.24.563710

Figure Lengend Snippet: a, Schematic of lipid delivery assay. 1) Incubation of empty P116 with fluorescent donor liposomes. 2) Removal of donor liposomes by ultracentrifugation and verification of removal by mass spectrometry. 3) Incubation of now fluorescently filled P116 with non-fluorescent DPPC acceptor liposomes. 4) Removal of P116 by ultrafiltration and verification of removal by dot blot, followed by analysis of fluorescent traits of acceptor liposomes by laser-scanning microscopy. b, Representative confocal light microscopy images. 1) Delivery into DPPC liposomes. 2) Delivery into HaCaT cells. (Left) Liposomes/cells (auto-fluorescence control), (middle) sample from the same workflow without P116 (spontaneous fusion/transfer control), and (right) sample with P116, according to the workflow detailed in a (lipid transfer with P116). The Dansyl and NBD channels are shown separately because of the large difference in excitation maxima and resulting use of separate laser lines. Both fluorophores emit in the green spectrum (at 527 nm). The NBD fluorescence signal is shown in red to facilitate interpretation of the merged image. c, Fluorescent lipids used in donor liposomes with their excitation and emission maxima. d, Statistical analysis of fluorescence differences between the sample in the presence of P116 and the controls. All differences are statistically significant. The experiment was carried out in triplicate, and all results could be replicated. The 25–75% data range is contained in the box, the horizontal line represents the median, the vertical line represents range within 1.5 interquartile range, the X represents the mean, and the circle represents outlier. Data can be found in Extended Data Table 1.

Article Snippet: The extracellular domain of P116, C-terminally shortened and HIS-tagged (30– 957), was expressed via the vector pOPINE_P116 backbone #26043; Addgene, Watertown, USA).

Techniques: Incubation, Liposomes, Mass Spectrometry, Dot Blot, Laser-Scanning Microscopy, Light Microscopy, Fluorescence

Journal: bioRxiv

Article Title: P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

doi: 10.1101/2023.10.24.563710

Figure Lengend Snippet: ab, Mass spectra show that compounds are specifically bound by P116. Compounds bound to P116 (blue), to P110 or P140 (other mycoplasma membrane proteins, black) or to the filter (red). a’b’, Structures of (a) a PROTAC and (b) a hydrophobic peptide that were used to load P116. Peaks corresponding to each compound are annotated in bold in the respective spectrum. c, The growth of cultured mycoplasma cells is inhibited by the tested compounds. Growth curves of M. pneumoniae treated with compound b’ (red), compound a’ (blue) and DMSO (black) in PBS at 35 h growth for 30 min before re-culturing in medium. The time of compound treatment is indicated with a black arrow. The experiment was carried out in triplicate, and all results could be replicated. Error bars represent standard deviation. d, Single-particle cryo-EM structure of P116 filled with hydrophobic peptide b’. The grey surface shows the density of peptide-filled P116 at 3.3 Å. It was fitted with the structure of P116 in the filled state (colored ribbon model, PDB: 8A9A). The surface representation in the lower panels is colored by hydrophobicity factor (yellow is hydrophobic and blue is hydrophilic) with the sectioning surface in grey. The peptide inside the hydrophobic cavity of P116 is colored red.

Article Snippet: The extracellular domain of P116, C-terminally shortened and HIS-tagged (30– 957), was expressed via the vector pOPINE_P116 backbone #26043; Addgene, Watertown, USA).

Techniques: Membrane, Cell Culture, Standard Deviation, Single Particle, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

doi: 10.1101/2023.10.24.563710

Figure Lengend Snippet: When empty, P116 is flexible enough to dorsally dock one monomer while distally docking the second monomer. The dorsal docking happens first, as it is based on long-range electrostatic interactions, which brings the second monomer close enough to the membrane to insert several phenylalanine residues (shown in yellow). Lipids enter P116 through the DCA (left) . Lipids inside the cavity are not shown. After the second monomer is filled, lipid head groups bind to residues in the dimerization domain, reducing the overall flexibility and possibly causing the monomer to detach (middle) . We propose that the filled monomer can now dock with the mycoplasma membrane and deliver its lipids through the DCA, following the concentration gradient (right) .

Article Snippet: The extracellular domain of P116, C-terminally shortened and HIS-tagged (30– 957), was expressed via the vector pOPINE_P116 backbone #26043; Addgene, Watertown, USA).

Techniques: Membrane, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes

doi: 10.1523/JNEUROSCI.2014-22.2023

Figure Lengend Snippet: Drift correction and localization error of QD probes in fixed hippocampal neurons expressing the YFP-tagged GluN1-1a subunit. A , Representative images of hippocampal neurons. Left, Neurons were stained by incubation in 20 n m MitoTracker Deep Red FM marker (catalog #M22426, Thermo Fisher Scientific) for 30 s in Neurobasal medium. Right, Neurons were infected with a lentivirus expressing the synaptic protein tdTomato-Homer1c. B , Schematic diagram depicting the three QD-based probes used in this study. The antiGFP-QD605 probe contains the rabbit anti-GFP IgG antibody combined with a secondary IgG antibody conjugated to QD605. The nanoGFP-QD605 probe contains an anti-GFP nanobody conjugated to QD605, while the nanoGFP-QD525 probe contains the anti-GFP nanobody conjugated to QD525. C , Negatively stained samples of both nanoGFP-QD probes were imaged at a magnification of 60,000× and a pixel size of 1.939 Å/px by TEM. Measured average diameters of both nanoGFP-QD probes ± SEM were 16.2 ± 0.4 nm (nanoGFP-QD525) and 20.4 ± 0.6 nm (nanoGFP-QD605; n ≥ 30). D , Example of drift estimated from multiple QD trajectories of the YFP-GluN1-1a subunit. E , The corresponding xy drift path. F , Example of raw QD trajectories (gray) and the drift-corrected QD trajectories (red) obtained by subtracting the drift path. G , Example images of fixed hippocampal neurons expressing the synaptic marker tdTomato-Homer1c (background pixels) and the YFP-GluN1-1a subunit labeled and tracked with the indicated QD-based probes (red). H , Scatter plots of all QD localizations; the red “+” indicates the mean value in both axes. I , Histograms showing the distribution of the distances between each QD localization shown in H ; the data were fitted with a Gaussian function, and the corresponding sigma (σ) values are indicated. J , Box plot summarizing the Gaussian fits performed on fixed QDs ( n = 20/group); one-way ANOVA F (2,57) = 121.84, p < 0.0001 followed by Bonferroni's multiple-comparisons test with p -values denoted in the figure. The average (mean ± SEM) localization errors were σ = 6.62 ± 0.25 nm (antiGFP-QD605), σ = 6.98 ± 0.32 nm (nanoGFP-QD605), and 15.34 ± 0.66 nm (nanoGFP-QD525).

Article Snippet: The bacterial expression vector encoding GFP nanobody (nanoGFP; catalog #49172, Addgene) was a gift from Brett Collins (The Institute for Molecular Bioscience, Queensland).

Techniques: Expressing, Staining, Incubation, Marker, Infection, Labeling