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    Name:
    GDP Solid
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    Catalog Number:
    NU-1172-1G
    Price:
    415.6
    Category:
    Nucleotides Nucleosides
    Size:
    1 g
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    Structured Review

    Jena Bioscience gdp
    Gsp1 interface mutations act allosterically to modulate the rate of <t>GTP</t> hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of <t>GDP</t> bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.

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    Images

    1) Product Images from "Biophysical basis of cellular multi-specificity encoded in a model molecular switch"

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch

    Journal: bioRxiv

    doi: 10.1101/2020.01.04.893909

    Gsp1 interface mutations act allosterically to modulate the rate of GTP hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of GDP bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.
    Figure Legend Snippet: Gsp1 interface mutations act allosterically to modulate the rate of GTP hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of GDP bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.

    Techniques Used: Nuclear Magnetic Resonance, Relative Rate, Mutagenesis, Standard Deviation, Sequencing

    Genetic interaction (GI) profiles of Gsp1 interface point mutants cluster by biological processes but not by targeted interfaces. a , Schematic summary of approach combining systems-level and biophysical measurements to characterize functional multi-specificity of a biological switch motif. b , Interface point mutations enable probing of the biological functions of the multi-specific GTPase switch Gsp1. c , Structures of Ran/Gsp1 in the GTP-bound (marine, PDB ID: 1ibr) and GDP-bound (gray, PDB ID: 3gj0) states. Mutated Gsp1 residues are shown as spheres. Switch loops I and II are shown in green and yellow, respectively. d , GI profiles of 23 Gsp1 mutants with nine or more significant GIs. Negative S-score (blue) represents synthetic sick/lethal GIs, positive S-score (yellow) represents suppressive/epistatic GIs. Mutants and genes are hierarchically clustered by Pearson correlation. e , Locations of mutated residues in structurally characterized interfaces. ΔrASA is the difference in accessible surface area of a residue upon binding, relative to an empirical maximum for the solvent accessible surface area of each amino acid residue type computed as in ( 5 ). f , Distributions of significant (see Methods) GIs of Gsp1 point mutants compared to GIs of mutant alleles of essential and non-essential genes. Red bars indicate the mean. g , Distributions of Pearson correlations between the GI profiles of Gsp1 interaction partners and Gsp1 mutants if mutation is (right, black) or is not (left, gray) in the interface with that partner. Point size indicates the false discovery rate adjusted one-sided (positive) p-value of the Pearson correlation. Red dots and bars indicate the mean and the upper and lower quartile, respectively.
    Figure Legend Snippet: Genetic interaction (GI) profiles of Gsp1 interface point mutants cluster by biological processes but not by targeted interfaces. a , Schematic summary of approach combining systems-level and biophysical measurements to characterize functional multi-specificity of a biological switch motif. b , Interface point mutations enable probing of the biological functions of the multi-specific GTPase switch Gsp1. c , Structures of Ran/Gsp1 in the GTP-bound (marine, PDB ID: 1ibr) and GDP-bound (gray, PDB ID: 3gj0) states. Mutated Gsp1 residues are shown as spheres. Switch loops I and II are shown in green and yellow, respectively. d , GI profiles of 23 Gsp1 mutants with nine or more significant GIs. Negative S-score (blue) represents synthetic sick/lethal GIs, positive S-score (yellow) represents suppressive/epistatic GIs. Mutants and genes are hierarchically clustered by Pearson correlation. e , Locations of mutated residues in structurally characterized interfaces. ΔrASA is the difference in accessible surface area of a residue upon binding, relative to an empirical maximum for the solvent accessible surface area of each amino acid residue type computed as in ( 5 ). f , Distributions of significant (see Methods) GIs of Gsp1 point mutants compared to GIs of mutant alleles of essential and non-essential genes. Red bars indicate the mean. g , Distributions of Pearson correlations between the GI profiles of Gsp1 interaction partners and Gsp1 mutants if mutation is (right, black) or is not (left, gray) in the interface with that partner. Point size indicates the false discovery rate adjusted one-sided (positive) p-value of the Pearson correlation. Red dots and bars indicate the mean and the upper and lower quartile, respectively.

    Techniques Used: Functional Assay, Binding Assay, Mutagenesis

    2) Product Images from "Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism *"

    Article Title: Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.651190

    Gα i1 is switched on by the exchange of GDP for GTP ( k off / k on ), then GTP hydrolysis proceeds ( k hyd ) and P i is released. Multiple turnover kinetics were measured via HPLC, which cannot distinguish between the three processes. Nucleotide exchange kinetics ( k off / k on ) were monitored via tryptophan fluorescence spectroscopy. Single turnover kinetics ( k hyd ) were measured via time-resolved FTIR difference spectroscopy.
    Figure Legend Snippet: Gα i1 is switched on by the exchange of GDP for GTP ( k off / k on ), then GTP hydrolysis proceeds ( k hyd ) and P i is released. Multiple turnover kinetics were measured via HPLC, which cannot distinguish between the three processes. Nucleotide exchange kinetics ( k off / k on ) were monitored via tryptophan fluorescence spectroscopy. Single turnover kinetics ( k hyd ) were measured via time-resolved FTIR difference spectroscopy.

    Techniques Used: High Performance Liquid Chromatography, Fluorescence, Spectroscopy

    3) Product Images from "Biophysical basis of cellular multi-specificity encoded in a model molecular switch"

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch

    Journal: bioRxiv

    doi: 10.1101/2020.01.04.893909

    Gsp1 interface mutations act allosterically to modulate the rate of GTP hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of GDP bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.
    Figure Legend Snippet: Gsp1 interface mutations act allosterically to modulate the rate of GTP hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of GDP bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.

    Techniques Used: Nuclear Magnetic Resonance, Relative Rate, Mutagenesis, Standard Deviation, Sequencing

    Genetic interaction (GI) profiles of Gsp1 interface point mutants cluster by biological processes but not by targeted interfaces. a , Schematic summary of approach combining systems-level and biophysical measurements to characterize functional multi-specificity of a biological switch motif. b , Interface point mutations enable probing of the biological functions of the multi-specific GTPase switch Gsp1. c , Structures of Ran/Gsp1 in the GTP-bound (marine, PDB ID: 1ibr) and GDP-bound (gray, PDB ID: 3gj0) states. Mutated Gsp1 residues are shown as spheres. Switch loops I and II are shown in green and yellow, respectively. d , GI profiles of 23 Gsp1 mutants with nine or more significant GIs. Negative S-score (blue) represents synthetic sick/lethal GIs, positive S-score (yellow) represents suppressive/epistatic GIs. Mutants and genes are hierarchically clustered by Pearson correlation. e , Locations of mutated residues in structurally characterized interfaces. ΔrASA is the difference in accessible surface area of a residue upon binding, relative to an empirical maximum for the solvent accessible surface area of each amino acid residue type computed as in ( 5 ). f , Distributions of significant (see Methods) GIs of Gsp1 point mutants compared to GIs of mutant alleles of essential and non-essential genes. Red bars indicate the mean. g , Distributions of Pearson correlations between the GI profiles of Gsp1 interaction partners and Gsp1 mutants if mutation is (right, black) or is not (left, gray) in the interface with that partner. Point size indicates the false discovery rate adjusted one-sided (positive) p-value of the Pearson correlation. Red dots and bars indicate the mean and the upper and lower quartile, respectively.
    Figure Legend Snippet: Genetic interaction (GI) profiles of Gsp1 interface point mutants cluster by biological processes but not by targeted interfaces. a , Schematic summary of approach combining systems-level and biophysical measurements to characterize functional multi-specificity of a biological switch motif. b , Interface point mutations enable probing of the biological functions of the multi-specific GTPase switch Gsp1. c , Structures of Ran/Gsp1 in the GTP-bound (marine, PDB ID: 1ibr) and GDP-bound (gray, PDB ID: 3gj0) states. Mutated Gsp1 residues are shown as spheres. Switch loops I and II are shown in green and yellow, respectively. d , GI profiles of 23 Gsp1 mutants with nine or more significant GIs. Negative S-score (blue) represents synthetic sick/lethal GIs, positive S-score (yellow) represents suppressive/epistatic GIs. Mutants and genes are hierarchically clustered by Pearson correlation. e , Locations of mutated residues in structurally characterized interfaces. ΔrASA is the difference in accessible surface area of a residue upon binding, relative to an empirical maximum for the solvent accessible surface area of each amino acid residue type computed as in ( 5 ). f , Distributions of significant (see Methods) GIs of Gsp1 point mutants compared to GIs of mutant alleles of essential and non-essential genes. Red bars indicate the mean. g , Distributions of Pearson correlations between the GI profiles of Gsp1 interaction partners and Gsp1 mutants if mutation is (right, black) or is not (left, gray) in the interface with that partner. Point size indicates the false discovery rate adjusted one-sided (positive) p-value of the Pearson correlation. Red dots and bars indicate the mean and the upper and lower quartile, respectively.

    Techniques Used: Functional Assay, Binding Assay, Mutagenesis

    4) Product Images from "Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1"

    Article Title: Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0102425

    Nucleotide-independent extraction of Rac1 Ic from the liposomes by GDI1. ( A ) GST-GDI1 pull-down of Rac1 Ic but not of Rac1 Ec . Input is the total mixture of beads and proteins, and output is the pull-down (PD). ( B ) Liposome binding of Rac1 Ic but not of Rac1 Ec . In the liposome sedimentation assay, Rac1 Ic efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1 Ec failed to bind to liposomes under the same conditions. ( C ) Preferential binding Rac1 Ic to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1 Ic proteins and prevents their association with the liposomes. ( D , E ) GDI1 efficiently extracted GDP-bound Rac1 Ic from the liposomes and to a lower extend also Rac1 Ic -GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1 Ic associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x g ( D ). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1 Ic -GppNHp from the liposomes to supernatants in comparison to Rac1 Ic -GDP ( E ). CBB, coomassie brilliant blue; Ec , E. coli ; Ic, insect cells; P, liposome pellet; S, supernatant.
    Figure Legend Snippet: Nucleotide-independent extraction of Rac1 Ic from the liposomes by GDI1. ( A ) GST-GDI1 pull-down of Rac1 Ic but not of Rac1 Ec . Input is the total mixture of beads and proteins, and output is the pull-down (PD). ( B ) Liposome binding of Rac1 Ic but not of Rac1 Ec . In the liposome sedimentation assay, Rac1 Ic efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1 Ec failed to bind to liposomes under the same conditions. ( C ) Preferential binding Rac1 Ic to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1 Ic proteins and prevents their association with the liposomes. ( D , E ) GDI1 efficiently extracted GDP-bound Rac1 Ic from the liposomes and to a lower extend also Rac1 Ic -GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1 Ic associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x g ( D ). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1 Ic -GppNHp from the liposomes to supernatants in comparison to Rac1 Ic -GDP ( E ). CBB, coomassie brilliant blue; Ec , E. coli ; Ic, insect cells; P, liposome pellet; S, supernatant.

    Techniques Used: Binding Assay, Sedimentation, Incubation

    5) Product Images from "Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein *"

    Article Title: Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.626275

    Binding of mant-deoxyguanine nucleotides to EFL1. Mant fluorescence was excited by FRET from the intrinsic tryptophan residues in EFL1. Concentrations after mixing consisted of 4 μ m EFL1, 50 μ m mant-deoxy-GDP/deoxy-GTP, and 5 m m Mg 2+ as
    Figure Legend Snippet: Binding of mant-deoxyguanine nucleotides to EFL1. Mant fluorescence was excited by FRET from the intrinsic tryptophan residues in EFL1. Concentrations after mixing consisted of 4 μ m EFL1, 50 μ m mant-deoxy-GDP/deoxy-GTP, and 5 m m Mg 2+ as

    Techniques Used: Binding Assay, Fluorescence

    Binding Kinetics of EFL1 to GDP and GTP
    Figure Legend Snippet: Binding Kinetics of EFL1 to GDP and GTP

    Techniques Used: Binding Assay

    6) Product Images from "The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway"

    Article Title: The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017816

    Dmknδ interacts with active and inactive Rab5. A. After capture of GST-Dmknδ5 fusion protein or GST alone on glutathione-sepharose beads, purified recombinant wild-type Rab5 (Rab5), incubated beforehand with GppNHp (active conformation) or GDP (inactive conformation), was loaded. Proteins initially loaded onto the column (input) or eluted from the column (output) were detected by immunoblotting with an antibody directed against Rab5. B. HeLa cells were transiently transfected with GFP-Dmknδ5 (green) and DsRed-Rab5Q79L or DsRed-Rab5S34N (red), respectively. Cells were then visualized by confocal microscopy. Bars, 5 µm
    Figure Legend Snippet: Dmknδ interacts with active and inactive Rab5. A. After capture of GST-Dmknδ5 fusion protein or GST alone on glutathione-sepharose beads, purified recombinant wild-type Rab5 (Rab5), incubated beforehand with GppNHp (active conformation) or GDP (inactive conformation), was loaded. Proteins initially loaded onto the column (input) or eluted from the column (output) were detected by immunoblotting with an antibody directed against Rab5. B. HeLa cells were transiently transfected with GFP-Dmknδ5 (green) and DsRed-Rab5Q79L or DsRed-Rab5S34N (red), respectively. Cells were then visualized by confocal microscopy. Bars, 5 µm

    Techniques Used: Purification, Recombinant, Incubation, Transfection, Confocal Microscopy

    Dmknδ expression in HeLa cells modifies the balance Rab5-GTP/Rab5-GDP. A . HeLa cells transiently transfected with DsRed-Rab5wt (red) and GFP-Dmknδ5 or GFP alone (green), were observed by confocal microscopy. Bars, 5 µm B. DsRed-Rab5wt was co-expressed in HeLa cells with GFP, GFP-Dmknδ5 (GFP-δ5), GFP-Dmknδ5-Nt (GFP-δ5Nt), or GFP-Dmknδ5-Ct (GFP-δ5Ct) as indicated. Top, detection of DsRed-Rab5wt-GTP amount retained by R5BD-GST with the anti-Rab5 antibody. Middle , total amount of DsRed-Rab5wt protein present in each HeLa protein extract used for the pull-down experiment as determined by immunoblot with the anti-Rab5 antibody. Bottom , expression analysis of the different GFP-tagged constructs immunoblotted with the anti-GFP antibody. C. Quantification of active DsRed-Rab5wt-GTP. The ratio of DsRed-Rab5wt-GTP over total DsRed-Rab5wt was determined for each condition. Western blots from two independent experiments were analysed by densitometry. Values are mean ± s.e.m.
    Figure Legend Snippet: Dmknδ expression in HeLa cells modifies the balance Rab5-GTP/Rab5-GDP. A . HeLa cells transiently transfected with DsRed-Rab5wt (red) and GFP-Dmknδ5 or GFP alone (green), were observed by confocal microscopy. Bars, 5 µm B. DsRed-Rab5wt was co-expressed in HeLa cells with GFP, GFP-Dmknδ5 (GFP-δ5), GFP-Dmknδ5-Nt (GFP-δ5Nt), or GFP-Dmknδ5-Ct (GFP-δ5Ct) as indicated. Top, detection of DsRed-Rab5wt-GTP amount retained by R5BD-GST with the anti-Rab5 antibody. Middle , total amount of DsRed-Rab5wt protein present in each HeLa protein extract used for the pull-down experiment as determined by immunoblot with the anti-Rab5 antibody. Bottom , expression analysis of the different GFP-tagged constructs immunoblotted with the anti-GFP antibody. C. Quantification of active DsRed-Rab5wt-GTP. The ratio of DsRed-Rab5wt-GTP over total DsRed-Rab5wt was determined for each condition. Western blots from two independent experiments were analysed by densitometry. Values are mean ± s.e.m.

    Techniques Used: Expressing, Transfection, Confocal Microscopy, Construct, Western Blot

    7) Product Images from "Directional transition from initiation to elongation in bacterial translation"

    Article Title: Directional transition from initiation to elongation in bacterial translation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv869

    Effect of GTP hydrolysis on binding of IF1 and IF2 to mature 70S IC. ( A ) 30S IC formed in the presence of Bpy-Met-tRNA fMet and GTP (12.5 μM) was rapidly mixed with 50S subunits in the presence or absence of GTPγS (0.25 mM). Time courses of Bpy-Met-tRNA fMet fluorescence changes were monitored. ( B ) Interaction of Bpy-Met-tRNA fMet with IF2 upon binding of the factor to 70S IC was followed by mixing purified 70S ICs (containing Bpy-Met-tRNA fMet ) with IF2 bound to GTPγS, GDPNP, GTP or GDP. Similar experiments were performed using an IF2 variant lacking the C2-domain (ΔC2) in the presence of GTPγS. ( C ) 30S S13 (Alx488) IC formed with IF1 4 (Atto540Q) and GTP (12.5 μM) was mixed with 50S subunits in the presence or absence of GTPγS (0.25 mM). ( D ) The binding of the IF1 to mature 70S IC was followed by mixing non-purified 70S ICs (formed with 30S S13 (Alx488) in the absence of IF1) with IF1 4 (Atto540Q), in the presence of GTP or GTPγS.
    Figure Legend Snippet: Effect of GTP hydrolysis on binding of IF1 and IF2 to mature 70S IC. ( A ) 30S IC formed in the presence of Bpy-Met-tRNA fMet and GTP (12.5 μM) was rapidly mixed with 50S subunits in the presence or absence of GTPγS (0.25 mM). Time courses of Bpy-Met-tRNA fMet fluorescence changes were monitored. ( B ) Interaction of Bpy-Met-tRNA fMet with IF2 upon binding of the factor to 70S IC was followed by mixing purified 70S ICs (containing Bpy-Met-tRNA fMet ) with IF2 bound to GTPγS, GDPNP, GTP or GDP. Similar experiments were performed using an IF2 variant lacking the C2-domain (ΔC2) in the presence of GTPγS. ( C ) 30S S13 (Alx488) IC formed with IF1 4 (Atto540Q) and GTP (12.5 μM) was mixed with 50S subunits in the presence or absence of GTPγS (0.25 mM). ( D ) The binding of the IF1 to mature 70S IC was followed by mixing non-purified 70S ICs (formed with 30S S13 (Alx488) in the absence of IF1) with IF1 4 (Atto540Q), in the presence of GTP or GTPγS.

    Techniques Used: Binding Assay, Fluorescence, Purification, Variant Assay

    Detailed kinetic scheme of late events in bacterial translation initiation. IF1, IF2–GTP, IF3, mRNA and fMet-tRNA fMet bind the 30S subunit to form a 30S IC. Step 1: Association of the 50S subunit to 30S IC to form a 70S PIC. Step 2: GTPase activation and rapid GTP hydrolysis ( 19 , 20 , 23 , 32 ). Step 3: Change of IF1 environment. Step 4: Pi release from IF2. Step 5: Release of the 3′ end of fMet-tRNA fMet from IF2 C2-domain. Step 6: Release of IF2 from the 70S complex and GDP from IF2; release of IF1 from the 70S complex, giving rise to an elongation competent 70S IC. Step 7: Binding of EF-Tu–GTP–aminoacyl-tRNA (TC) to the 70S IC is followed by peptide bond formation to form a 70S EC. Dissociation of IF3 from the 70S complex was reported to proceed at an apparent rate of 3.2 s −1 ( 21 ); because IF3 may undergo conformational changes and movements while the complex maturates, the position of IF3 is indicated by a lighter shade and should be considered tentative. Dissociation of IF1 and IF2 to 70S IC, as well as step 5, become reversible in the absence of GTP hydrolysis, as indicated by dashed arrows. The observables employed to monitor each step are summarized in Table 2 .
    Figure Legend Snippet: Detailed kinetic scheme of late events in bacterial translation initiation. IF1, IF2–GTP, IF3, mRNA and fMet-tRNA fMet bind the 30S subunit to form a 30S IC. Step 1: Association of the 50S subunit to 30S IC to form a 70S PIC. Step 2: GTPase activation and rapid GTP hydrolysis ( 19 , 20 , 23 , 32 ). Step 3: Change of IF1 environment. Step 4: Pi release from IF2. Step 5: Release of the 3′ end of fMet-tRNA fMet from IF2 C2-domain. Step 6: Release of IF2 from the 70S complex and GDP from IF2; release of IF1 from the 70S complex, giving rise to an elongation competent 70S IC. Step 7: Binding of EF-Tu–GTP–aminoacyl-tRNA (TC) to the 70S IC is followed by peptide bond formation to form a 70S EC. Dissociation of IF3 from the 70S complex was reported to proceed at an apparent rate of 3.2 s −1 ( 21 ); because IF3 may undergo conformational changes and movements while the complex maturates, the position of IF3 is indicated by a lighter shade and should be considered tentative. Dissociation of IF1 and IF2 to 70S IC, as well as step 5, become reversible in the absence of GTP hydrolysis, as indicated by dashed arrows. The observables employed to monitor each step are summarized in Table 2 .

    Techniques Used: Activation Assay, Binding Assay

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    other:

    Article Title: Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein *
    Article Snippet: The fluorescent mant-derivatives of GDP, GTP, dGDP, dGTP, and GppNHp were obtained from Jena Biosciences (Jena, Germany).

    Article Title: Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1
    Article Snippet: GDP and a non-hydrolyzable GTP analogue, guanosine 5′-[β,γ-imido]triphosphate (GppNHp), were obtained from Jena Bioscience GmbH (Jena, Germany).

    Variant Assay:

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch
    Article Snippet: Kinetic measurements of Srm1 mediated nucleotide exchange Kinetic parameters of GEF mediated nucleotide exchange were determined using a fluorescence resonance energy transfer (FRET) based protocol ( ). .. Each Gsp1 variant was purified as a Gsp1:GDP complex, as confirmed by reverse phase chromatography. .. Nucleotide exchange from GDP to mant-GTP (2’-(or-3’)-O-(N-Methylanthraniloyl) Guanosine 5-Triphosphate, CAT # NU-206L, Jena Biosciences) was monitored by measuring a decrease in intrinsic Gsp1 tryptophan fluorescence (295 nm excitation, 335 nm detection) due to FRET upon binding of the mant group.

    Purification:

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch
    Article Snippet: Kinetic measurements of Srm1 mediated nucleotide exchange Kinetic parameters of GEF mediated nucleotide exchange were determined using a fluorescence resonance energy transfer (FRET) based protocol ( ). .. Each Gsp1 variant was purified as a Gsp1:GDP complex, as confirmed by reverse phase chromatography. .. Nucleotide exchange from GDP to mant-GTP (2’-(or-3’)-O-(N-Methylanthraniloyl) Guanosine 5-Triphosphate, CAT # NU-206L, Jena Biosciences) was monitored by measuring a decrease in intrinsic Gsp1 tryptophan fluorescence (295 nm excitation, 335 nm detection) due to FRET upon binding of the mant group.

    Reversed-phase Chromatography:

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch
    Article Snippet: Kinetic measurements of Srm1 mediated nucleotide exchange Kinetic parameters of GEF mediated nucleotide exchange were determined using a fluorescence resonance energy transfer (FRET) based protocol ( ). .. Each Gsp1 variant was purified as a Gsp1:GDP complex, as confirmed by reverse phase chromatography. .. Nucleotide exchange from GDP to mant-GTP (2’-(or-3’)-O-(N-Methylanthraniloyl) Guanosine 5-Triphosphate, CAT # NU-206L, Jena Biosciences) was monitored by measuring a decrease in intrinsic Gsp1 tryptophan fluorescence (295 nm excitation, 335 nm detection) due to FRET upon binding of the mant group.

    Recombinant:

    Article Title: The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway
    Article Snippet: .. In some cases, cleaved Rab5bwt recombinant protein was preloaded with 500 µM of GppNHp, a non-hydrolysable analogue of GTP or 500 µM of GDP (Jena Biosciences), overnight at 4°C, in the presence of 10 mM EDTA and 0.3% β-mercaptoethanol. .. The nucleotide binding reaction was stopped by adding 10 mM MgCl2 .

    Fluorescence:

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch
    Article Snippet: Each Gsp1 variant was purified as a Gsp1:GDP complex, as confirmed by reverse phase chromatography. .. Nucleotide exchange from GDP to mant-GTP (2’-(or-3’)-O-(N-Methylanthraniloyl) Guanosine 5-Triphosphate, CAT # NU-206L, Jena Biosciences) was monitored by measuring a decrease in intrinsic Gsp1 tryptophan fluorescence (295 nm excitation, 335 nm detection) due to FRET upon binding of the mant group. .. Each time course was measured in GEF assay buffer (40 mM HEPES pH 7.5, 100 mM NaCl, 4 mM MgCl2 , 1 mM Dithiothreitol) with excess of mant-GTP.

    Binding Assay:

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch
    Article Snippet: Each Gsp1 variant was purified as a Gsp1:GDP complex, as confirmed by reverse phase chromatography. .. Nucleotide exchange from GDP to mant-GTP (2’-(or-3’)-O-(N-Methylanthraniloyl) Guanosine 5-Triphosphate, CAT # NU-206L, Jena Biosciences) was monitored by measuring a decrease in intrinsic Gsp1 tryptophan fluorescence (295 nm excitation, 335 nm detection) due to FRET upon binding of the mant group. .. Each time course was measured in GEF assay buffer (40 mM HEPES pH 7.5, 100 mM NaCl, 4 mM MgCl2 , 1 mM Dithiothreitol) with excess of mant-GTP.

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    Jena Bioscience gdp
    Gsp1 interface mutations act allosterically to modulate the rate of <t>GTP</t> hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of <t>GDP</t> bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.
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    Gsp1 interface mutations act allosterically to modulate the rate of GTP hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of GDP bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.

    Journal: bioRxiv

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch

    doi: 10.1101/2020.01.04.893909

    Figure Lengend Snippet: Gsp1 interface mutations act allosterically to modulate the rate of GTP hydrolysis. a , Annotated 1D 31 P NMR spectrum of wild-type Gsp1 loaded with GTP. Peak areas are computed over intervals shown and normalized to the GTPβ bound (GTPβbound) peak. The peaks from left to right correspond to: free phosphate (Pi), β phosphate of GDP bound to Gsp1 (GDPβbound), β phosphate of free (unbound) GDP (GDPβfree), γ phosphate of GTP bound to Gsp1 in conformation 1 (γ1), γ phosphate of GTP bound to Gsp1 in conformation 2 (γ2), α phosphate of bound or unbound GDP or GTP, β phosphate of GTP bound to Gsp1 (GTPβbound), β phosphate of free (unbound) GTP (GTPβfree). b , Rate of intrinsic GTP hydrolysis of wild-type Gsp1 and mutants. Dotted line indicates wild-type value. Error bars represent the standard deviations from n 3 replicates (except for A180T which has two replicates). c , Percent population in γ2 state plotted against the relative rate of intrinsic GTP hydrolysis represented as a natural logarithm of the ratio of the rate for the mutant over the rate of the wild type. The pink line is a linear fit. Error bars represent the standard deviation from n 3 replicates of intrinsic GTP hydrolysis measurements (except for A180T which has two replicates). d , Structures of HRas (in cartoon representation, blue) bound to allosteric inhibitors shown in stick representation: BI 2852 (PDB ID: 6gj8, light violet), vinylsulfonamide (PDB ID: 4m1w, dark violet), and AMG 510 (PDB ID: 6oim, deepsalmon). Switch I and switch II regions of HRas are in green and yellow, respectively. Human HRas residues corresponding to Gsp1 allosteric sites (identified from the sequence alignment between Gsp1 and human HRas) are represented as pink spheres. The corresponding Gsp1 residues are in parentheses.

    Article Snippet: Nucleotide exchange from GDP to mant-GTP (2’-(or-3’)-O-(N-Methylanthraniloyl) Guanosine 5-Triphosphate, CAT # NU-206L, Jena Biosciences) was monitored by measuring a decrease in intrinsic Gsp1 tryptophan fluorescence (295 nm excitation, 335 nm detection) due to FRET upon binding of the mant group.

    Techniques: Nuclear Magnetic Resonance, Relative Rate, Mutagenesis, Standard Deviation, Sequencing

    Genetic interaction (GI) profiles of Gsp1 interface point mutants cluster by biological processes but not by targeted interfaces. a , Schematic summary of approach combining systems-level and biophysical measurements to characterize functional multi-specificity of a biological switch motif. b , Interface point mutations enable probing of the biological functions of the multi-specific GTPase switch Gsp1. c , Structures of Ran/Gsp1 in the GTP-bound (marine, PDB ID: 1ibr) and GDP-bound (gray, PDB ID: 3gj0) states. Mutated Gsp1 residues are shown as spheres. Switch loops I and II are shown in green and yellow, respectively. d , GI profiles of 23 Gsp1 mutants with nine or more significant GIs. Negative S-score (blue) represents synthetic sick/lethal GIs, positive S-score (yellow) represents suppressive/epistatic GIs. Mutants and genes are hierarchically clustered by Pearson correlation. e , Locations of mutated residues in structurally characterized interfaces. ΔrASA is the difference in accessible surface area of a residue upon binding, relative to an empirical maximum for the solvent accessible surface area of each amino acid residue type computed as in ( 5 ). f , Distributions of significant (see Methods) GIs of Gsp1 point mutants compared to GIs of mutant alleles of essential and non-essential genes. Red bars indicate the mean. g , Distributions of Pearson correlations between the GI profiles of Gsp1 interaction partners and Gsp1 mutants if mutation is (right, black) or is not (left, gray) in the interface with that partner. Point size indicates the false discovery rate adjusted one-sided (positive) p-value of the Pearson correlation. Red dots and bars indicate the mean and the upper and lower quartile, respectively.

    Journal: bioRxiv

    Article Title: Biophysical basis of cellular multi-specificity encoded in a model molecular switch

    doi: 10.1101/2020.01.04.893909

    Figure Lengend Snippet: Genetic interaction (GI) profiles of Gsp1 interface point mutants cluster by biological processes but not by targeted interfaces. a , Schematic summary of approach combining systems-level and biophysical measurements to characterize functional multi-specificity of a biological switch motif. b , Interface point mutations enable probing of the biological functions of the multi-specific GTPase switch Gsp1. c , Structures of Ran/Gsp1 in the GTP-bound (marine, PDB ID: 1ibr) and GDP-bound (gray, PDB ID: 3gj0) states. Mutated Gsp1 residues are shown as spheres. Switch loops I and II are shown in green and yellow, respectively. d , GI profiles of 23 Gsp1 mutants with nine or more significant GIs. Negative S-score (blue) represents synthetic sick/lethal GIs, positive S-score (yellow) represents suppressive/epistatic GIs. Mutants and genes are hierarchically clustered by Pearson correlation. e , Locations of mutated residues in structurally characterized interfaces. ΔrASA is the difference in accessible surface area of a residue upon binding, relative to an empirical maximum for the solvent accessible surface area of each amino acid residue type computed as in ( 5 ). f , Distributions of significant (see Methods) GIs of Gsp1 point mutants compared to GIs of mutant alleles of essential and non-essential genes. Red bars indicate the mean. g , Distributions of Pearson correlations between the GI profiles of Gsp1 interaction partners and Gsp1 mutants if mutation is (right, black) or is not (left, gray) in the interface with that partner. Point size indicates the false discovery rate adjusted one-sided (positive) p-value of the Pearson correlation. Red dots and bars indicate the mean and the upper and lower quartile, respectively.

    Article Snippet: Nucleotide exchange from GDP to mant-GTP (2’-(or-3’)-O-(N-Methylanthraniloyl) Guanosine 5-Triphosphate, CAT # NU-206L, Jena Biosciences) was monitored by measuring a decrease in intrinsic Gsp1 tryptophan fluorescence (295 nm excitation, 335 nm detection) due to FRET upon binding of the mant group.

    Techniques: Functional Assay, Binding Assay, Mutagenesis

    Gα i1 is switched on by the exchange of GDP for GTP ( k off / k on ), then GTP hydrolysis proceeds ( k hyd ) and P i is released. Multiple turnover kinetics were measured via HPLC, which cannot distinguish between the three processes. Nucleotide exchange kinetics ( k off / k on ) were monitored via tryptophan fluorescence spectroscopy. Single turnover kinetics ( k hyd ) were measured via time-resolved FTIR difference spectroscopy.

    Journal: The Journal of Biological Chemistry

    Article Title: Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism *

    doi: 10.1074/jbc.M115.651190

    Figure Lengend Snippet: Gα i1 is switched on by the exchange of GDP for GTP ( k off / k on ), then GTP hydrolysis proceeds ( k hyd ) and P i is released. Multiple turnover kinetics were measured via HPLC, which cannot distinguish between the three processes. Nucleotide exchange kinetics ( k off / k on ) were monitored via tryptophan fluorescence spectroscopy. Single turnover kinetics ( k hyd ) were measured via time-resolved FTIR difference spectroscopy.

    Article Snippet: Lyophilized GDP, GTP, and GTPγS were purchased from Jena Bioscience (Jena, Germany).

    Techniques: High Performance Liquid Chromatography, Fluorescence, Spectroscopy

    Nucleotide-independent extraction of Rac1 Ic from the liposomes by GDI1. ( A ) GST-GDI1 pull-down of Rac1 Ic but not of Rac1 Ec . Input is the total mixture of beads and proteins, and output is the pull-down (PD). ( B ) Liposome binding of Rac1 Ic but not of Rac1 Ec . In the liposome sedimentation assay, Rac1 Ic efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1 Ec failed to bind to liposomes under the same conditions. ( C ) Preferential binding Rac1 Ic to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1 Ic proteins and prevents their association with the liposomes. ( D , E ) GDI1 efficiently extracted GDP-bound Rac1 Ic from the liposomes and to a lower extend also Rac1 Ic -GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1 Ic associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x g ( D ). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1 Ic -GppNHp from the liposomes to supernatants in comparison to Rac1 Ic -GDP ( E ). CBB, coomassie brilliant blue; Ec , E. coli ; Ic, insect cells; P, liposome pellet; S, supernatant.

    Journal: PLoS ONE

    Article Title: Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

    doi: 10.1371/journal.pone.0102425

    Figure Lengend Snippet: Nucleotide-independent extraction of Rac1 Ic from the liposomes by GDI1. ( A ) GST-GDI1 pull-down of Rac1 Ic but not of Rac1 Ec . Input is the total mixture of beads and proteins, and output is the pull-down (PD). ( B ) Liposome binding of Rac1 Ic but not of Rac1 Ec . In the liposome sedimentation assay, Rac1 Ic efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1 Ec failed to bind to liposomes under the same conditions. ( C ) Preferential binding Rac1 Ic to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1 Ic proteins and prevents their association with the liposomes. ( D , E ) GDI1 efficiently extracted GDP-bound Rac1 Ic from the liposomes and to a lower extend also Rac1 Ic -GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1 Ic associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x g ( D ). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1 Ic -GppNHp from the liposomes to supernatants in comparison to Rac1 Ic -GDP ( E ). CBB, coomassie brilliant blue; Ec , E. coli ; Ic, insect cells; P, liposome pellet; S, supernatant.

    Article Snippet: GDP and a non-hydrolyzable GTP analogue, guanosine 5′-[β,γ-imido]triphosphate (GppNHp), were obtained from Jena Bioscience GmbH (Jena, Germany).

    Techniques: Binding Assay, Sedimentation, Incubation