Lipins are magnesium-dependent phosphatidic acid phosphatases that catalyze the dephosphorylation of phosphatidic acid to generate diacylglycerol. Lipins play a major role in lipid homeostasis, including de novo phospholipid biogenesis, fat storage as triglycerides, adipocyte differentiation, and lipoprotein secretion. Mutations in lipins are known to cause diseases such as rhabdomyolysis, insulin resistance, and Majeed syndrome. Lipins are the only enzymes in the major pathway of triglyceride synthesis that are not constitutively membrane bound but associate with membranes transiently to act upon their substrate. For this reason, we are interested in the structure of lipins and more specifically, how lipins recognize their hydrophobic, membrane bound substrate to understand how disease mutations result in loss of function. To determine this, we plan to solve the crystal structure of a minimal, enzymatically active version of this enzyme since much of its structure is intrinsically disordered. We plan to co- crystallize this construct with magnesium, short-chain water soluble lipid substrates and products, or other substrate analogs. To better understand substrate preference and how membrane-packing defects affect lipin function, we tested the activity and membrane interaction of lipins with lipids of different fatty acyl chain composition and with liposomes of different lipid compositions. We will also determine the structural and biochemical impact of the rhabdomyolysis mutation with in vitro assays as well as structural studies. Together, these data will help to better understand regulation of lipin activity and possibly in the future, help to provide treatments to the diseases caused by lipin dysfunction. This work was supported by a Gilliam Fellowship from the Howard Hughes Medical Institute as well as a grant from NIGMS (R35 GM128666).
Data associated with the publication of the same title for single-molecule FRET experiments used to characterize conformational ensembles of the PSG supramodule of PSD-95. Folder 1. Single Molecule Experimental Data .ht3 and .ptu files corresponding to time-tagged photon records collected using single-photon detectors with Hydra Harp TCSPC software and hardware. Folders are provided for each sample. Selection criteria and correction parameters are detailed in the manuscript. DO data were taken from bursts with SPIE<0.1 while AO data were SPIE>0.9. 1.1 BG Background files from which background signals were calculated for each sample. 1.2 IRF IRF data files used for generation of IRF curves for fluorescence decay histogram analysis and generation of fFCS filters. Fluorescence Decay Histograms Data files for photon counts in each time bin following donor excitation pulse for Donor-only (DO), FRET (DA), and Instrument Response Function (IRF) curves used in fluorescence decay histogram analysis. Filtered FCS Curves and Filter Files Data corresponding to four fFCS curves per sample: low-FRET-high-FRET (lohi), high-FRET-low-FRET (hilo), low-FRET-low-FRET (lolo), and high-FRET-high-FRET (hihi), as well as filter files generated in the Kristine software package using the .ht3 and .ptu data and IRF files from Single Molecule Experimental Data. Simulation AV-Derived Distance Values Structures from DMD simulations and AV-derived interdye distances used for structural classification against experimental FRET data. Also includes references files for associating distances correctly with structures and FRET pairs.
Green fluorescent protein (GFP)-like fluorescent proteins have been found in more than 120 species. Although the proteins have little sequence identity, Gly31, 33, and 35 are 87, 100, and 95% conserved across all species, respectively. All GFP-like proteins have a β-barrel structure composed of 11 β-sheets, and the 3 conserved glycines are located in the second β-sheet. Molecular dynamics (MD) simulations have shown that mutating one or more of the glycines to alanines most likely does not reduce chromophore formation in correctly folded immature fluorescent proteins. MD and protein characterization of alanine mutants indicate that mutation of the conserved glycines leads to misfolding. Gly31, 33, and 35 are essential to maintain the integrity of the β1-3 triad that is the last structural element to slot in place in the formation of the canonical fluorescent protein β-barrel. Glycines located in β-sheets may have a similar role in the formation of other non-GFP β-barrels.
This file details the folder structure for the data files provided corresponding to the experiments and analysis performed in Fuzzy Supertertiary Interactions within PSD-95 Enable Ligand Binding. Correction parameters, results, and methods are detailed in the manuscript. The file “Sample Reference.xlsx” details the naming conventions for different FRET labeling site variants. “RefitDistanceTables.xlsx” provides the distances for all global fits of subsets of samples performed for classification of DMD structures. The .zip file "PDBDev_adtl_Datasets.zip" Contains additional files corresponding to submissions of structures from the corresponding manuscript to wwPDB-Dev. Folder 1. Single Molecule Experimental Data .ht3 and .ptu files corresponding to time-tagged photon records collected using single-photon detectors with Hydra Harp TCSPC software and hardware. Folders are provided for each sample. Selection criteria and correction parameters are detailed in the manuscript. DO data were taken from bursts with SPIE<0.1 while AO data were SPIE>0.9. 1.1 BG Background files from which background signals were calculated for each sample. Subfolder for each sample. 1.2 IRF IRF data files used for generation of IRF curves for fluorescence decay histogram analysis and generation of fFCS filters. Subfolder for each sample. Fluorescence Decay Histograms Data files for photon counts in each time bin following donor excitation pulse for Donor-only (DO), FRET (DA), and Instrument Response Function (IRF) curves used in fluorescence decay histogram analysis. 2.1 Fit Curves Curves fit to decay histograms, in xyxy format (time delay after pulse, fit curve counts, time, residual) Filtered FCS Curves and Filter Files Data corresponding to four fFCS curves per sample: low-FRET-high-FRET (lohi), high-FRET-low-FRET (hilo), low-FRET-low-FRET (lolo), and high-FRET-high-FRET (hihi), as well as filter files generated in the Kristine software package using the .ht3 and .ptu data and IRF files from Single Molecule Experimental Data. 3.1 Fits Fit curves and residuals for all samples. .cor files are simple text files in xy format (third column unused). .res, .fit files follow likewise. Simulation AV-Derived Distance Values Structures from DMD simulations and AV-derived interdye distances used for structural classification against experimental FRET data. Also includes references files for associating distances correctly with structures and FRET pairs. 4.1 CoM AV Contours and PCA Data Files contain AV-derived distance information for all simulation structures from DMD, in compact npy format loadable via numpy.load. The text files contain center-of-mass positions in xyz format for the indicated domains. 5. MFD Histograms Contains text files for MFD contour plot data, as well as FRET line equations for each sample. Text files are in matrix representation, with the first row and column being the x/y axis values and all internal values representing a 2D bin. 6. PDA Histograms Contains time-binned histograms of smFRET data and modeling outputs from PDA fitting.
This file details the folder structure for the data files provided corresponding to the experiments and analysis performed in Fuzzy Supertertiary Interactions within PSD-95 Enable Ligand Binding. Correction parameters, results, and methods are detailed in the manuscript. The file “Sample Reference.xlsx” details the naming conventions for different FRET labeling site variants. “RefitDistanceTables.xlsx” provides the distances for all global fits of subsets of samples performed for classification of DMD structures. The .zip file "PDBDev_adtl_Datasets.zip" Contains additional files corresponding to submissions of structures from the corresponding manuscript to wwPDB-Dev. Folder 1. Single Molecule Experimental Data .ht3 and .ptu files corresponding to time-tagged photon records collected using single-photon detectors with Hydra Harp TCSPC software and hardware. Folders are provided for each sample. Selection criteria and correction parameters are detailed in the manuscript. DO data were taken from bursts with SPIE<0.1 while AO data were SPIE>0.9. 1.1 BG Background files from which background signals were calculated for each sample. Subfolder for each sample. 1.2 IRF IRF data files used for generation of IRF curves for fluorescence decay histogram analysis and generation of fFCS filters. Subfolder for each sample. Fluorescence Decay Histograms Data files for photon counts in each time bin following donor excitation pulse for Donor-only (DO), FRET (DA), and Instrument Response Function (IRF) curves used in fluorescence decay histogram analysis. 2.1 Fit Curves Curves fit to decay histograms, in xyxy format (time delay after pulse, fit curve counts, time, residual) Filtered FCS Curves and Filter Files Data corresponding to four fFCS curves per sample: low-FRET-high-FRET (lohi), high-FRET-low-FRET (hilo), low-FRET-low-FRET (lolo), and high-FRET-high-FRET (hihi), as well as filter files generated in the Kristine software package using the .ht3 and .ptu data and IRF files from Single Molecule Experimental Data. 3.1 Fits Fit curves and residuals for all samples. .cor files are simple text files in xy format (third column unused). .res, .fit files follow likewise. Simulation AV-Derived Distance Values Structures from DMD simulations and AV-derived interdye distances used for structural classification against experimental FRET data. Also includes references files for associating distances correctly with structures and FRET pairs. 4.1 CoM AV Contours and PCA Data Files contain AV-derived distance information for all simulation structures from DMD, in compact npy format loadable via numpy.load. The text files contain center-of-mass positions in xyz format for the indicated domains. 5. MFD Histograms Contains text files for MFD contour plot data, as well as FRET line equations for each sample. Text files are in matrix representation, with the first row and column being the x/y axis values and all internal values representing a 2D bin. 6. PDA Histograms Contains time-binned histograms of smFRET data and modeling outputs from PDA fitting.
This file details the folder structure for the data files provided corresponding to the experiments and analysis performed in Fuzzy Supertertiary Interactions within PSD-95 Enable Ligand Binding. Correction parameters, results, and methods are detailed in the manuscript. The file “Sample Reference.xlsx” details the naming conventions for different FRET labeling site variants. “RefitDistanceTables.xlsx” provides the distances for all global fits of subsets of samples performed for classification of DMD structures. Folder 1. Single Molecule Experimental Data .ht3 and .ptu files corresponding to time-tagged photon records collected using single-photon detectors with Hydra Harp TCSPC software and hardware. Folders are provided for each sample. Selection criteria and correction parameters are detailed in the manuscript. DO data were taken from bursts with SPIE<0.1 while AO data were SPIE>0.9. 1.1 BG Background files from which background signals were calculated for each sample. Subfolder for each sample. 1.2 IRF IRF data files used for generation of IRF curves for fluorescence decay histogram analysis and generation of fFCS filters. Subfolder for each sample. Fluorescence Decay Histograms Data files for photon counts in each time bin following donor excitation pulse for Donor-only (DO), FRET (DA), and Instrument Response Function (IRF) curves used in fluorescence decay histogram analysis. 2.1 Fit Curves Curves fit to decay histograms, in xyxy format (time delay after pulse, fit curve counts, time, residual) Filtered FCS Curves and Filter Files Data corresponding to four fFCS curves per sample: low-FRET-high-FRET (lohi), high-FRET-low-FRET (hilo), low-FRET-low-FRET (lolo), and high-FRET-high-FRET (hihi), as well as filter files generated in the Kristine software package using the .ht3 and .ptu data and IRF files from Single Molecule Experimental Data. 3.1 Fits Fit curves and residuals for all samples. .cor files are simple text files in xy format (third column unused). .res, .fit files follow likewise. Simulation AV-Derived Distance Values Structures from DMD simulations and AV-derived interdye distances used for structural classification against experimental FRET data. Also includes references files for associating distances correctly with structures and FRET pairs. 4.1 CoM AV Contours and PCA Data Files contain AV-derived distance information for all simulation structures from DMD, in compact npy format loadable via numpy.load. The text files contain center-of-mass positions in xyz format for the indicated domains. 5. MFD Histograms Contains text files for MFD contour plot data, as well as FRET line equations for each sample. Text files are in matrix representation, with the first row and column being the x/y axis values and all internal values representing a 2D bin. 6. PDA Histograms Contains time-binned histograms of smFRET data and modeling outputs from PDA fitting.
This file details the folder structure for the data files provided corresponding to the experiments and analysis performed in Fuzzy Supertertiary Interactions within PSD-95 Enable Ligand Binding. Correction parameters, results, and methods are detailed in the manuscript. The file “Sample Reference.xlsx” details the naming conventions for different FRET labeling site variants. “RefitDistanceTables.xlsx” provides the distances for all global fits of subsets of samples performed for classification of DMD structures. The .zip file "PDBDev_adtl_Datasets.zip" Contains additional files corresponding to submissions of structures from the corresponding manuscript to wwPDB-Dev. Folder 1. Single Molecule Experimental Data .ht3 and .ptu files corresponding to time-tagged photon records collected using single-photon detectors with Hydra Harp TCSPC software and hardware. Folders are provided for each sample. Selection criteria and correction parameters are detailed in the manuscript. DO data were taken from bursts with SPIE<0.1 while AO data were SPIE>0.9. 1.1 BG Background files from which background signals were calculated for each sample. Subfolder for each sample. 1.2 IRF IRF data files used for generation of IRF curves for fluorescence decay histogram analysis and generation of fFCS filters. Subfolder for each sample. Fluorescence Decay Histograms Data files for photon counts in each time bin following donor excitation pulse for Donor-only (DO), FRET (DA), and Instrument Response Function (IRF) curves used in fluorescence decay histogram analysis. 2.1 Fit Curves Curves fit to decay histograms, in xyxy format (time delay after pulse, fit curve counts, time, residual) Filtered FCS Curves and Filter Files Data corresponding to four fFCS curves per sample: low-FRET-high-FRET (lohi), high-FRET-low-FRET (hilo), low-FRET-low-FRET (lolo), and high-FRET-high-FRET (hihi), as well as filter files generated in the Kristine software package using the .ht3 and .ptu data and IRF files from Single Molecule Experimental Data. 3.1 Fits Fit curves and residuals for all samples. .cor files are simple text files in xy format (third column unused). .res, .fit files follow likewise. Simulation AV-Derived Distance Values Structures from DMD simulations and AV-derived interdye distances used for structural classification against experimental FRET data. Also includes references files for associating distances correctly with structures and FRET pairs. 4.1 CoM AV Contours and PCA Data Files contain AV-derived distance information for all simulation structures from DMD, in compact npy format loadable via numpy.load. The text files contain center-of-mass positions in xyz format for the indicated domains. 5. MFD Histograms Contains text files for MFD contour plot data, as well as FRET line equations for each sample. Text files are in matrix representation, with the first row and column being the x/y axis values and all internal values representing a 2D bin. 6. PDA Histograms Contains time-binned histograms of smFRET data and modeling outputs from PDA fitting.
ABSTRACT The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by PDZ3, SH3 and GuK domains, regulate PSD-95 activity. Here we combined Discrete Molecular Dynamics and single molecule FRET to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand.
