mant atpγs  (Jena Bioscience)


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    Name:
    Mant ATPγS
    Description:

    Catalog Number:
    nu-232l
    Price:
    352.14
    Applications:
    Inhibition of heart adenylyl cyclase AC5[1] Inhibition of adenylyl cyclase from Bordetella pertussis[2] FRET[2]
    Purity:
    ≥ 90 % (HPLC)
    Category:
    Nucleotides Nucleosides
    Buy from Supplier


    Structured Review

    Jena Bioscience mant atpγs
    PAN's conserved arginines are not involved in regulating ATP-binding affinity. Equilibrium ATP-binding affinity was determined by monitoring the change in fluorescence intensity of <t>mant-ATPγS</t> (15 nM) in the presence of increasing amounts of WT-PAN, PAN-R328A, PAN-R331A or PAN-R328/331A. The x axis is concentration of binding sites considering two high-affinity binding sites per PAN hexamer. The Michaelis–Menten binding hyperbola was fit to the raw data using nonlinear regression analysis to obtain the K d (inset); the quality of fit ( R 2 ) is also shown.

    https://www.bioz.com/result/mant atpγs/product/Jena Bioscience
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mant atpγs - by Bioz Stars, 2020-07
    92/100 stars

    Images

    1) Product Images from "ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function"

    Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

    Journal: Nature Communications

    doi: 10.1038/ncomms9520

    PAN's conserved arginines are not involved in regulating ATP-binding affinity. Equilibrium ATP-binding affinity was determined by monitoring the change in fluorescence intensity of mant-ATPγS (15 nM) in the presence of increasing amounts of WT-PAN, PAN-R328A, PAN-R331A or PAN-R328/331A. The x axis is concentration of binding sites considering two high-affinity binding sites per PAN hexamer. The Michaelis–Menten binding hyperbola was fit to the raw data using nonlinear regression analysis to obtain the K d (inset); the quality of fit ( R 2 ) is also shown.
    Figure Legend Snippet: PAN's conserved arginines are not involved in regulating ATP-binding affinity. Equilibrium ATP-binding affinity was determined by monitoring the change in fluorescence intensity of mant-ATPγS (15 nM) in the presence of increasing amounts of WT-PAN, PAN-R328A, PAN-R331A or PAN-R328/331A. The x axis is concentration of binding sites considering two high-affinity binding sites per PAN hexamer. The Michaelis–Menten binding hyperbola was fit to the raw data using nonlinear regression analysis to obtain the K d (inset); the quality of fit ( R 2 ) is also shown.

    Techniques Used: Binding Assay, Fluorescence, Concentration Assay

    Both of the conserved arginines in PAN are required for ATP hydrolysis, but not for ATP binding. ( a ) Specific ATPase activity of WT and arginine mutants of PAN in the presence of 1 mM ATP. ATPase rates were determined with a real-time assay. ( b ) m-ATPγS (15 nM) binds to PAN and the arginine mutants (1 μM). Nucleotide binding is evident by a change in the intensity of mant fluorescence upon binding to PAN. Representative data are presented from three independent experiments ±s.d.
    Figure Legend Snippet: Both of the conserved arginines in PAN are required for ATP hydrolysis, but not for ATP binding. ( a ) Specific ATPase activity of WT and arginine mutants of PAN in the presence of 1 mM ATP. ATPase rates were determined with a real-time assay. ( b ) m-ATPγS (15 nM) binds to PAN and the arginine mutants (1 μM). Nucleotide binding is evident by a change in the intensity of mant fluorescence upon binding to PAN. Representative data are presented from three independent experiments ±s.d.

    Techniques Used: Binding Assay, Activity Assay, Fluorescence

    Mutation of either one of PAN's conserved arginines abrogates ATP-dependent substrate binding and 20S gate opening. ( a ) Fluorescence polarization was used to monitor the binding of GFP–ssrA (0.08 μM) to PAN (0.12 μM) or its arginine mutants in the presence of 1 mM ADP (negative control) or 1 mM ATPγS. ( b ) Gate opening in the 20S proteasome (20 nM) by PAN WT, or its mutants (80 nM), was monitored with the LFP peptide hydrolysis in the presence of 10 μM ATPγS. ‘No PAN' is 20S (archaeal) alone. ( c ) Gate opening in the 20S proteasome (20 nM) as a function of increasing concentration of WT-PAN and arginine mutants. ( d ) The gate-opening assay by the WT-PAN (10 nM) as in b but also in the presence of the other indicated PAN mutants (10 nM) to determine whether the mutants can compete with WT for binding to the 20S. All data are representative experiments and are the means of three independent measurements ±s.d.
    Figure Legend Snippet: Mutation of either one of PAN's conserved arginines abrogates ATP-dependent substrate binding and 20S gate opening. ( a ) Fluorescence polarization was used to monitor the binding of GFP–ssrA (0.08 μM) to PAN (0.12 μM) or its arginine mutants in the presence of 1 mM ADP (negative control) or 1 mM ATPγS. ( b ) Gate opening in the 20S proteasome (20 nM) by PAN WT, or its mutants (80 nM), was monitored with the LFP peptide hydrolysis in the presence of 10 μM ATPγS. ‘No PAN' is 20S (archaeal) alone. ( c ) Gate opening in the 20S proteasome (20 nM) as a function of increasing concentration of WT-PAN and arginine mutants. ( d ) The gate-opening assay by the WT-PAN (10 nM) as in b but also in the presence of the other indicated PAN mutants (10 nM) to determine whether the mutants can compete with WT for binding to the 20S. All data are representative experiments and are the means of three independent measurements ±s.d.

    Techniques Used: Mutagenesis, Binding Assay, Fluorescence, Negative Control, Concentration Assay

    PAN's conserved arginines are not involved in regulating nucleotide stoichiometry, or the ATP-binding pattern. ( a ) The number of m-ATPγS that bound to PAN (90 nM) was determined by rapid separation of bound nucleotide from free nucleotide using 100 μl spin columns at two different concentrations of ATPγS: 10 and 200 μM. Ten micromolar saturates only the two high-affinity sites, and 200 μM allows near saturation of the high- and low-affinity sites (ATP and ADP sites; ref. 8 ). The number of bound nucleotides per PAN hexamer was calculated for WT and each arginine mutant as labelled. Data are means of four independent experiments ±s.d. ( b ) Emission spectra of m-ATP as in Fig. 1a , but with PAN-R328/331A (1 μM). Quantifications are presented on Table 1 . ( c , d ) The number of m-ATPγS-bound nucleotides to the labelled PAN variant was calculated as in a at increasing nucleotide concentrations to generate a binding curve. [PAN] was 200 nM and thus the free ligand bind approximation is not met here and thus the K -value is expressed as Kobs as it does not accurately quantify affinity. Representative data are presented from three independent experiments ±s.d.
    Figure Legend Snippet: PAN's conserved arginines are not involved in regulating nucleotide stoichiometry, or the ATP-binding pattern. ( a ) The number of m-ATPγS that bound to PAN (90 nM) was determined by rapid separation of bound nucleotide from free nucleotide using 100 μl spin columns at two different concentrations of ATPγS: 10 and 200 μM. Ten micromolar saturates only the two high-affinity sites, and 200 μM allows near saturation of the high- and low-affinity sites (ATP and ADP sites; ref. 8 ). The number of bound nucleotides per PAN hexamer was calculated for WT and each arginine mutant as labelled. Data are means of four independent experiments ±s.d. ( b ) Emission spectra of m-ATP as in Fig. 1a , but with PAN-R328/331A (1 μM). Quantifications are presented on Table 1 . ( c , d ) The number of m-ATPγS-bound nucleotides to the labelled PAN variant was calculated as in a at increasing nucleotide concentrations to generate a binding curve. [PAN] was 200 nM and thus the free ligand bind approximation is not met here and thus the K -value is expressed as Kobs as it does not accurately quantify affinity. Representative data are presented from three independent experiments ±s.d.

    Techniques Used: Binding Assay, Mutagenesis, Variant Assay

    ATP and ADP off-rates are similar and the ADP off-rate is not affected by mutation of the arginine finger. ( a ) Pre-steady-state dissociation of the prebound m-ADP (150 nM) from WT-PAN (150 nM) was monitored by stopped-flow at 37 °C. Saturating amounts ADP (2 mM) were used to compete off the m-ADP. The residuals from fitting the raw data with single- or double-exponential decay models are shown (right). ( b ) The half-life ( T 1/2 ) of the bound m-ADP to WT-PAN and the arginine mutants for the double-decay model is presented, showing both fast and slow rates. ( c ) Pre-steady state dissociation of prebound m-ATPγS (1 μM) from WT-PAN (0.5 μM) was monitored as in a . Saturating amounts ADP (4 mM) were used to compete off the m-ATPγS. Residuals for the single- and double-decay models are shown (right). The determined half-life for both fast and slow rates for m-ATPγS are shown in the inset (double-decay model).
    Figure Legend Snippet: ATP and ADP off-rates are similar and the ADP off-rate is not affected by mutation of the arginine finger. ( a ) Pre-steady-state dissociation of the prebound m-ADP (150 nM) from WT-PAN (150 nM) was monitored by stopped-flow at 37 °C. Saturating amounts ADP (2 mM) were used to compete off the m-ADP. The residuals from fitting the raw data with single- or double-exponential decay models are shown (right). ( b ) The half-life ( T 1/2 ) of the bound m-ADP to WT-PAN and the arginine mutants for the double-decay model is presented, showing both fast and slow rates. ( c ) Pre-steady state dissociation of prebound m-ATPγS (1 μM) from WT-PAN (0.5 μM) was monitored as in a . Saturating amounts ADP (4 mM) were used to compete off the m-ATPγS. Residuals for the single- and double-decay models are shown (right). The determined half-life for both fast and slow rates for m-ATPγS are shown in the inset (double-decay model).

    Techniques Used: Mutagenesis, Flow Cytometry

    ATP binds to neighbouring subunits (‘ortho' pattern) in the archaeal and mammalian proteasomal ATPases. ( a ) Emission spectra of m-ATP (1 μM) in the presence or absence of the indicated nucleotides (1 μM each) and PAN-E271Q (1 μM) at equilibrium (37 °C). The FRET (with t-ATP) and no-FRET conditions are shown and are colour labelled. ( b ) Same conditions as in a but with m-ATPγS and WT-PAN at 4 °C. ( c ) Same conditions as in b but with bovine 26S proteasome (1 μM) instead of PAN. ( d ) Same conditions as in a , but with the addition of GFP–ssrA (1 μM), which was photobleached by ultraviolet treatment before the assay to minimize the inner filter effect of GFP. ( e ) Structure of the 26S proteasomal ATPases (4CR4—atomic model derived from an 8-Å cryo-EM map), indicating the distance measurements between the sensor-2 residues in the various nucleotide-binding sites ( o -ortho, m -meta and p -para). ( f ) Estimated average distance and ranges between ortho-, meta- and para-positioned nucleotide-binding sites in the eukaryotic 26S ATPases (4CR4) corresponding to e .
    Figure Legend Snippet: ATP binds to neighbouring subunits (‘ortho' pattern) in the archaeal and mammalian proteasomal ATPases. ( a ) Emission spectra of m-ATP (1 μM) in the presence or absence of the indicated nucleotides (1 μM each) and PAN-E271Q (1 μM) at equilibrium (37 °C). The FRET (with t-ATP) and no-FRET conditions are shown and are colour labelled. ( b ) Same conditions as in a but with m-ATPγS and WT-PAN at 4 °C. ( c ) Same conditions as in b but with bovine 26S proteasome (1 μM) instead of PAN. ( d ) Same conditions as in a , but with the addition of GFP–ssrA (1 μM), which was photobleached by ultraviolet treatment before the assay to minimize the inner filter effect of GFP. ( e ) Structure of the 26S proteasomal ATPases (4CR4—atomic model derived from an 8-Å cryo-EM map), indicating the distance measurements between the sensor-2 residues in the various nucleotide-binding sites ( o -ortho, m -meta and p -para). ( f ) Estimated average distance and ranges between ortho-, meta- and para-positioned nucleotide-binding sites in the eukaryotic 26S ATPases (4CR4) corresponding to e .

    Techniques Used: Derivative Assay, Binding Assay

    2) Product Images from "Asymmetric nucleotide transactions of the HslUV protease"

    Article Title: Asymmetric nucleotide transactions of the HslUV protease

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2008.05.070

    HslU binding to FL-gt1, a fluorescent substrate-mimic peptide. ( A ) Binding of FL-gt1 (100 nM) by wild-type HslU or E257Q HslU in the presence/absence of HslV 12 . Binding reactions contained 500 μM ATPγS (HslU) or 500 μM ATP (E257Q HslU) and were performed at 25 °C in PD buffer. The fitted lines are for non-cooperative binding with K d ’s of 0.76 μM (HslUV), 0.93 μM (HslU), 3.0 μM (E257Q HslUV), and 5.6 μM (E257Q). ( B ) Binding of E257Q HslU (1.5 μM hexamer) to FL-gt1 (0.1 μM) as a function of ATPγS concentration (top panel) or N6-methyl-ATP concentration (bottom panel). The data were fitted to the Hill equation. Half-maximal binding occurred at 0.8 μM ATPγS (n = 2.3 ± 0.23), 34 μM N6-methyl-ATP without HslV (n = 1.8 ± 0.15), and 2.3 μM N6-methyl-ATP with 3 μM HslV 12 (n = 1.5 ± 0.12).
    Figure Legend Snippet: HslU binding to FL-gt1, a fluorescent substrate-mimic peptide. ( A ) Binding of FL-gt1 (100 nM) by wild-type HslU or E257Q HslU in the presence/absence of HslV 12 . Binding reactions contained 500 μM ATPγS (HslU) or 500 μM ATP (E257Q HslU) and were performed at 25 °C in PD buffer. The fitted lines are for non-cooperative binding with K d ’s of 0.76 μM (HslUV), 0.93 μM (HslU), 3.0 μM (E257Q HslUV), and 5.6 μM (E257Q). ( B ) Binding of E257Q HslU (1.5 μM hexamer) to FL-gt1 (0.1 μM) as a function of ATPγS concentration (top panel) or N6-methyl-ATP concentration (bottom panel). The data were fitted to the Hill equation. Half-maximal binding occurred at 0.8 μM ATPγS (n = 2.3 ± 0.23), 34 μM N6-methyl-ATP without HslV (n = 1.8 ± 0.15), and 2.3 μM N6-methyl-ATP with 3 μM HslV 12 (n = 1.5 ± 0.12).

    Techniques Used: Binding Assay, Concentration Assay

    HslU·HslV binding. ( A ) Binding of HslU (top) or E257Q HslU (bottom) to HslV 12 (15 nM) was assayed by changes in the rate of Z-Gly-Gly-AMC peptide cleavage. Reactions contained 1 mM ATPγS or 1 mM ATP and were performed in PD buffer at 25 °C. Data were fit to a quadratic form of a hyperbolic binding isotherm. Apparent K D values were 12 ± 2 nM (HslU; ATPγS), 78 ± 10 nM (HslU; ATP), 19 ± 5 nM (E257Q HslU; ATP) and 21 ± 2 nM (E257Q HslU; ATPγS). ( B (10 μM Arc-IA37-st11-titin-ssrA) strengthened binding of HslV to HslU (0.3 μM hexamer), as measured by changes in the rate of HslU ATP hydrolysis. HslUV·Arc complexes were preassembled by incubating for 5 min with 10 μM ATPγS at 37 °C. Reactions in PD buffer at 37 °C were initiated by addition of ATP and a regeneration system. ( C ) HslU activation of HslV 12 (100 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM) was assayed in the presence of 100, 200, 400 or 800 nM ATPγS. ( D ) Rates of Z-Gly-Gly-Leu-AMC (200 μM) cleavage were assayed in the presence of 1 μM HslU hexamer, 100 nM ATPγS, and increasing HslV 12 . ( E ) N6-methyl-ATP supports hyperbolic activation of HslU 6 (50 nM) stimulation of HslV 12 (200 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM). The solid line is a fit (R = 0.997) to the equation activity = 100/(1+([0.85 μM][N6-methyl-ATP])).
    Figure Legend Snippet: HslU·HslV binding. ( A ) Binding of HslU (top) or E257Q HslU (bottom) to HslV 12 (15 nM) was assayed by changes in the rate of Z-Gly-Gly-AMC peptide cleavage. Reactions contained 1 mM ATPγS or 1 mM ATP and were performed in PD buffer at 25 °C. Data were fit to a quadratic form of a hyperbolic binding isotherm. Apparent K D values were 12 ± 2 nM (HslU; ATPγS), 78 ± 10 nM (HslU; ATP), 19 ± 5 nM (E257Q HslU; ATP) and 21 ± 2 nM (E257Q HslU; ATPγS). ( B (10 μM Arc-IA37-st11-titin-ssrA) strengthened binding of HslV to HslU (0.3 μM hexamer), as measured by changes in the rate of HslU ATP hydrolysis. HslUV·Arc complexes were preassembled by incubating for 5 min with 10 μM ATPγS at 37 °C. Reactions in PD buffer at 37 °C were initiated by addition of ATP and a regeneration system. ( C ) HslU activation of HslV 12 (100 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM) was assayed in the presence of 100, 200, 400 or 800 nM ATPγS. ( D ) Rates of Z-Gly-Gly-Leu-AMC (200 μM) cleavage were assayed in the presence of 1 μM HslU hexamer, 100 nM ATPγS, and increasing HslV 12 . ( E ) N6-methyl-ATP supports hyperbolic activation of HslU 6 (50 nM) stimulation of HslV 12 (200 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM). The solid line is a fit (R = 0.997) to the equation activity = 100/(1+([0.85 μM][N6-methyl-ATP])).

    Techniques Used: Binding Assay, Activation Assay, Activity Assay

    HslU. ( A ) Two HslU 6 ATPases (blue) can assemble with the HslV 12 peptidase (magenta). ( B ) HslU hexamers have six potential nucleotide binding sites, located at domain and subunit interfaces. From 3–6 nucleotides bind HslU 6 in different crystal structures. ( C (10 μM). Reactions components were preincubated with 10 μM ATPγS to promote HslU or HslUV association prior to addition of ATP.
    Figure Legend Snippet: HslU. ( A ) Two HslU 6 ATPases (blue) can assemble with the HslV 12 peptidase (magenta). ( B ) HslU hexamers have six potential nucleotide binding sites, located at domain and subunit interfaces. From 3–6 nucleotides bind HslU 6 in different crystal structures. ( C (10 μM). Reactions components were preincubated with 10 μM ATPγS to promote HslU or HslUV association prior to addition of ATP.

    Techniques Used: Binding Assay

    Related Articles

    Binding Assay:

    Article Title: Unified mechanisms for self-RNA recognition by RIG-I Singleton-Merten syndrome variants
    Article Snippet: .. Binding of MANT-ATP and MANT-ATPγS to different full-length RIG-I mutants (1–925, N-terminal His-tag) was determined via Förster resonance energy transfer from RIG-I to MANT-ATP (Jena Bioscience, Jena, Germany). .. All samples were prepared in 96-well black chimney microplates.

    Article Title: Unified mechanisms for self-RNA recognition by RIG-I Singleton-Merten syndrome variants
    Article Snippet: .. MANT-ATP binding Binding of MANT-ATP and MANT-ATPγS to different full-length RIG-I mutants (1–925, N-terminal His-tag) was determined via Förster resonance energy transfer from RIG-I to MANT-ATP (Jena Bioscience, Jena, Germany). .. All samples were prepared in 96-well black chimney microplates.

    Förster Resonance Energy Transfer:

    Article Title: Unified mechanisms for self-RNA recognition by RIG-I Singleton-Merten syndrome variants
    Article Snippet: .. Binding of MANT-ATP and MANT-ATPγS to different full-length RIG-I mutants (1–925, N-terminal His-tag) was determined via Förster resonance energy transfer from RIG-I to MANT-ATP (Jena Bioscience, Jena, Germany). .. All samples were prepared in 96-well black chimney microplates.

    Article Title: Unified mechanisms for self-RNA recognition by RIG-I Singleton-Merten syndrome variants
    Article Snippet: .. MANT-ATP binding Binding of MANT-ATP and MANT-ATPγS to different full-length RIG-I mutants (1–925, N-terminal His-tag) was determined via Förster resonance energy transfer from RIG-I to MANT-ATP (Jena Bioscience, Jena, Germany). .. All samples were prepared in 96-well black chimney microplates.

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    Jena Bioscience mant atpγs
    PAN's conserved arginines are not involved in regulating ATP-binding affinity. Equilibrium ATP-binding affinity was determined by monitoring the change in fluorescence intensity of <t>mant-ATPγS</t> (15 nM) in the presence of increasing amounts of WT-PAN, PAN-R328A, PAN-R331A or PAN-R328/331A. The x axis is concentration of binding sites considering two high-affinity binding sites per PAN hexamer. The Michaelis–Menten binding hyperbola was fit to the raw data using nonlinear regression analysis to obtain the K d (inset); the quality of fit ( R 2 ) is also shown.
    Mant Atpγs, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mant atpγs/product/Jena Bioscience
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mant atpγs - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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    PAN's conserved arginines are not involved in regulating ATP-binding affinity. Equilibrium ATP-binding affinity was determined by monitoring the change in fluorescence intensity of mant-ATPγS (15 nM) in the presence of increasing amounts of WT-PAN, PAN-R328A, PAN-R331A or PAN-R328/331A. The x axis is concentration of binding sites considering two high-affinity binding sites per PAN hexamer. The Michaelis–Menten binding hyperbola was fit to the raw data using nonlinear regression analysis to obtain the K d (inset); the quality of fit ( R 2 ) is also shown.

    Journal: Nature Communications

    Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

    doi: 10.1038/ncomms9520

    Figure Lengend Snippet: PAN's conserved arginines are not involved in regulating ATP-binding affinity. Equilibrium ATP-binding affinity was determined by monitoring the change in fluorescence intensity of mant-ATPγS (15 nM) in the presence of increasing amounts of WT-PAN, PAN-R328A, PAN-R331A or PAN-R328/331A. The x axis is concentration of binding sites considering two high-affinity binding sites per PAN hexamer. The Michaelis–Menten binding hyperbola was fit to the raw data using nonlinear regression analysis to obtain the K d (inset); the quality of fit ( R 2 ) is also shown.

    Article Snippet: Mant- ATPγS and Mant-ADP were purchased from Jena Bioscience.

    Techniques: Binding Assay, Fluorescence, Concentration Assay

    Both of the conserved arginines in PAN are required for ATP hydrolysis, but not for ATP binding. ( a ) Specific ATPase activity of WT and arginine mutants of PAN in the presence of 1 mM ATP. ATPase rates were determined with a real-time assay. ( b ) m-ATPγS (15 nM) binds to PAN and the arginine mutants (1 μM). Nucleotide binding is evident by a change in the intensity of mant fluorescence upon binding to PAN. Representative data are presented from three independent experiments ±s.d.

    Journal: Nature Communications

    Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

    doi: 10.1038/ncomms9520

    Figure Lengend Snippet: Both of the conserved arginines in PAN are required for ATP hydrolysis, but not for ATP binding. ( a ) Specific ATPase activity of WT and arginine mutants of PAN in the presence of 1 mM ATP. ATPase rates were determined with a real-time assay. ( b ) m-ATPγS (15 nM) binds to PAN and the arginine mutants (1 μM). Nucleotide binding is evident by a change in the intensity of mant fluorescence upon binding to PAN. Representative data are presented from three independent experiments ±s.d.

    Article Snippet: Mant- ATPγS and Mant-ADP were purchased from Jena Bioscience.

    Techniques: Binding Assay, Activity Assay, Fluorescence

    Mutation of either one of PAN's conserved arginines abrogates ATP-dependent substrate binding and 20S gate opening. ( a ) Fluorescence polarization was used to monitor the binding of GFP–ssrA (0.08 μM) to PAN (0.12 μM) or its arginine mutants in the presence of 1 mM ADP (negative control) or 1 mM ATPγS. ( b ) Gate opening in the 20S proteasome (20 nM) by PAN WT, or its mutants (80 nM), was monitored with the LFP peptide hydrolysis in the presence of 10 μM ATPγS. ‘No PAN' is 20S (archaeal) alone. ( c ) Gate opening in the 20S proteasome (20 nM) as a function of increasing concentration of WT-PAN and arginine mutants. ( d ) The gate-opening assay by the WT-PAN (10 nM) as in b but also in the presence of the other indicated PAN mutants (10 nM) to determine whether the mutants can compete with WT for binding to the 20S. All data are representative experiments and are the means of three independent measurements ±s.d.

    Journal: Nature Communications

    Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

    doi: 10.1038/ncomms9520

    Figure Lengend Snippet: Mutation of either one of PAN's conserved arginines abrogates ATP-dependent substrate binding and 20S gate opening. ( a ) Fluorescence polarization was used to monitor the binding of GFP–ssrA (0.08 μM) to PAN (0.12 μM) or its arginine mutants in the presence of 1 mM ADP (negative control) or 1 mM ATPγS. ( b ) Gate opening in the 20S proteasome (20 nM) by PAN WT, or its mutants (80 nM), was monitored with the LFP peptide hydrolysis in the presence of 10 μM ATPγS. ‘No PAN' is 20S (archaeal) alone. ( c ) Gate opening in the 20S proteasome (20 nM) as a function of increasing concentration of WT-PAN and arginine mutants. ( d ) The gate-opening assay by the WT-PAN (10 nM) as in b but also in the presence of the other indicated PAN mutants (10 nM) to determine whether the mutants can compete with WT for binding to the 20S. All data are representative experiments and are the means of three independent measurements ±s.d.

    Article Snippet: Mant- ATPγS and Mant-ADP were purchased from Jena Bioscience.

    Techniques: Mutagenesis, Binding Assay, Fluorescence, Negative Control, Concentration Assay

    PAN's conserved arginines are not involved in regulating nucleotide stoichiometry, or the ATP-binding pattern. ( a ) The number of m-ATPγS that bound to PAN (90 nM) was determined by rapid separation of bound nucleotide from free nucleotide using 100 μl spin columns at two different concentrations of ATPγS: 10 and 200 μM. Ten micromolar saturates only the two high-affinity sites, and 200 μM allows near saturation of the high- and low-affinity sites (ATP and ADP sites; ref. 8 ). The number of bound nucleotides per PAN hexamer was calculated for WT and each arginine mutant as labelled. Data are means of four independent experiments ±s.d. ( b ) Emission spectra of m-ATP as in Fig. 1a , but with PAN-R328/331A (1 μM). Quantifications are presented on Table 1 . ( c , d ) The number of m-ATPγS-bound nucleotides to the labelled PAN variant was calculated as in a at increasing nucleotide concentrations to generate a binding curve. [PAN] was 200 nM and thus the free ligand bind approximation is not met here and thus the K -value is expressed as Kobs as it does not accurately quantify affinity. Representative data are presented from three independent experiments ±s.d.

    Journal: Nature Communications

    Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

    doi: 10.1038/ncomms9520

    Figure Lengend Snippet: PAN's conserved arginines are not involved in regulating nucleotide stoichiometry, or the ATP-binding pattern. ( a ) The number of m-ATPγS that bound to PAN (90 nM) was determined by rapid separation of bound nucleotide from free nucleotide using 100 μl spin columns at two different concentrations of ATPγS: 10 and 200 μM. Ten micromolar saturates only the two high-affinity sites, and 200 μM allows near saturation of the high- and low-affinity sites (ATP and ADP sites; ref. 8 ). The number of bound nucleotides per PAN hexamer was calculated for WT and each arginine mutant as labelled. Data are means of four independent experiments ±s.d. ( b ) Emission spectra of m-ATP as in Fig. 1a , but with PAN-R328/331A (1 μM). Quantifications are presented on Table 1 . ( c , d ) The number of m-ATPγS-bound nucleotides to the labelled PAN variant was calculated as in a at increasing nucleotide concentrations to generate a binding curve. [PAN] was 200 nM and thus the free ligand bind approximation is not met here and thus the K -value is expressed as Kobs as it does not accurately quantify affinity. Representative data are presented from three independent experiments ±s.d.

    Article Snippet: Mant- ATPγS and Mant-ADP were purchased from Jena Bioscience.

    Techniques: Binding Assay, Mutagenesis, Variant Assay

    ATP and ADP off-rates are similar and the ADP off-rate is not affected by mutation of the arginine finger. ( a ) Pre-steady-state dissociation of the prebound m-ADP (150 nM) from WT-PAN (150 nM) was monitored by stopped-flow at 37 °C. Saturating amounts ADP (2 mM) were used to compete off the m-ADP. The residuals from fitting the raw data with single- or double-exponential decay models are shown (right). ( b ) The half-life ( T 1/2 ) of the bound m-ADP to WT-PAN and the arginine mutants for the double-decay model is presented, showing both fast and slow rates. ( c ) Pre-steady state dissociation of prebound m-ATPγS (1 μM) from WT-PAN (0.5 μM) was monitored as in a . Saturating amounts ADP (4 mM) were used to compete off the m-ATPγS. Residuals for the single- and double-decay models are shown (right). The determined half-life for both fast and slow rates for m-ATPγS are shown in the inset (double-decay model).

    Journal: Nature Communications

    Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

    doi: 10.1038/ncomms9520

    Figure Lengend Snippet: ATP and ADP off-rates are similar and the ADP off-rate is not affected by mutation of the arginine finger. ( a ) Pre-steady-state dissociation of the prebound m-ADP (150 nM) from WT-PAN (150 nM) was monitored by stopped-flow at 37 °C. Saturating amounts ADP (2 mM) were used to compete off the m-ADP. The residuals from fitting the raw data with single- or double-exponential decay models are shown (right). ( b ) The half-life ( T 1/2 ) of the bound m-ADP to WT-PAN and the arginine mutants for the double-decay model is presented, showing both fast and slow rates. ( c ) Pre-steady state dissociation of prebound m-ATPγS (1 μM) from WT-PAN (0.5 μM) was monitored as in a . Saturating amounts ADP (4 mM) were used to compete off the m-ATPγS. Residuals for the single- and double-decay models are shown (right). The determined half-life for both fast and slow rates for m-ATPγS are shown in the inset (double-decay model).

    Article Snippet: Mant- ATPγS and Mant-ADP were purchased from Jena Bioscience.

    Techniques: Mutagenesis, Flow Cytometry

    ATP binds to neighbouring subunits (‘ortho' pattern) in the archaeal and mammalian proteasomal ATPases. ( a ) Emission spectra of m-ATP (1 μM) in the presence or absence of the indicated nucleotides (1 μM each) and PAN-E271Q (1 μM) at equilibrium (37 °C). The FRET (with t-ATP) and no-FRET conditions are shown and are colour labelled. ( b ) Same conditions as in a but with m-ATPγS and WT-PAN at 4 °C. ( c ) Same conditions as in b but with bovine 26S proteasome (1 μM) instead of PAN. ( d ) Same conditions as in a , but with the addition of GFP–ssrA (1 μM), which was photobleached by ultraviolet treatment before the assay to minimize the inner filter effect of GFP. ( e ) Structure of the 26S proteasomal ATPases (4CR4—atomic model derived from an 8-Å cryo-EM map), indicating the distance measurements between the sensor-2 residues in the various nucleotide-binding sites ( o -ortho, m -meta and p -para). ( f ) Estimated average distance and ranges between ortho-, meta- and para-positioned nucleotide-binding sites in the eukaryotic 26S ATPases (4CR4) corresponding to e .

    Journal: Nature Communications

    Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

    doi: 10.1038/ncomms9520

    Figure Lengend Snippet: ATP binds to neighbouring subunits (‘ortho' pattern) in the archaeal and mammalian proteasomal ATPases. ( a ) Emission spectra of m-ATP (1 μM) in the presence or absence of the indicated nucleotides (1 μM each) and PAN-E271Q (1 μM) at equilibrium (37 °C). The FRET (with t-ATP) and no-FRET conditions are shown and are colour labelled. ( b ) Same conditions as in a but with m-ATPγS and WT-PAN at 4 °C. ( c ) Same conditions as in b but with bovine 26S proteasome (1 μM) instead of PAN. ( d ) Same conditions as in a , but with the addition of GFP–ssrA (1 μM), which was photobleached by ultraviolet treatment before the assay to minimize the inner filter effect of GFP. ( e ) Structure of the 26S proteasomal ATPases (4CR4—atomic model derived from an 8-Å cryo-EM map), indicating the distance measurements between the sensor-2 residues in the various nucleotide-binding sites ( o -ortho, m -meta and p -para). ( f ) Estimated average distance and ranges between ortho-, meta- and para-positioned nucleotide-binding sites in the eukaryotic 26S ATPases (4CR4) corresponding to e .

    Article Snippet: Mant- ATPγS and Mant-ADP were purchased from Jena Bioscience.

    Techniques: Derivative Assay, Binding Assay

    HslU binding to FL-gt1, a fluorescent substrate-mimic peptide. ( A ) Binding of FL-gt1 (100 nM) by wild-type HslU or E257Q HslU in the presence/absence of HslV 12 . Binding reactions contained 500 μM ATPγS (HslU) or 500 μM ATP (E257Q HslU) and were performed at 25 °C in PD buffer. The fitted lines are for non-cooperative binding with K d ’s of 0.76 μM (HslUV), 0.93 μM (HslU), 3.0 μM (E257Q HslUV), and 5.6 μM (E257Q). ( B ) Binding of E257Q HslU (1.5 μM hexamer) to FL-gt1 (0.1 μM) as a function of ATPγS concentration (top panel) or N6-methyl-ATP concentration (bottom panel). The data were fitted to the Hill equation. Half-maximal binding occurred at 0.8 μM ATPγS (n = 2.3 ± 0.23), 34 μM N6-methyl-ATP without HslV (n = 1.8 ± 0.15), and 2.3 μM N6-methyl-ATP with 3 μM HslV 12 (n = 1.5 ± 0.12).

    Journal: Journal of molecular biology

    Article Title: Asymmetric nucleotide transactions of the HslUV protease

    doi: 10.1016/j.jmb.2008.05.070

    Figure Lengend Snippet: HslU binding to FL-gt1, a fluorescent substrate-mimic peptide. ( A ) Binding of FL-gt1 (100 nM) by wild-type HslU or E257Q HslU in the presence/absence of HslV 12 . Binding reactions contained 500 μM ATPγS (HslU) or 500 μM ATP (E257Q HslU) and were performed at 25 °C in PD buffer. The fitted lines are for non-cooperative binding with K d ’s of 0.76 μM (HslUV), 0.93 μM (HslU), 3.0 μM (E257Q HslUV), and 5.6 μM (E257Q). ( B ) Binding of E257Q HslU (1.5 μM hexamer) to FL-gt1 (0.1 μM) as a function of ATPγS concentration (top panel) or N6-methyl-ATP concentration (bottom panel). The data were fitted to the Hill equation. Half-maximal binding occurred at 0.8 μM ATPγS (n = 2.3 ± 0.23), 34 μM N6-methyl-ATP without HslV (n = 1.8 ± 0.15), and 2.3 μM N6-methyl-ATP with 3 μM HslV 12 (n = 1.5 ± 0.12).

    Article Snippet: Triethylammonium salts of mant-ATP, mant-ATPγS, and ADP were purchased from Jena Biosciences GmbH (Jena, Germany); concentrations were determined by absorbance at 355 nm using an extinction coefficient of 5,800 M−1 cm−1 .

    Techniques: Binding Assay, Concentration Assay

    HslU·HslV binding. ( A ) Binding of HslU (top) or E257Q HslU (bottom) to HslV 12 (15 nM) was assayed by changes in the rate of Z-Gly-Gly-AMC peptide cleavage. Reactions contained 1 mM ATPγS or 1 mM ATP and were performed in PD buffer at 25 °C. Data were fit to a quadratic form of a hyperbolic binding isotherm. Apparent K D values were 12 ± 2 nM (HslU; ATPγS), 78 ± 10 nM (HslU; ATP), 19 ± 5 nM (E257Q HslU; ATP) and 21 ± 2 nM (E257Q HslU; ATPγS). ( B (10 μM Arc-IA37-st11-titin-ssrA) strengthened binding of HslV to HslU (0.3 μM hexamer), as measured by changes in the rate of HslU ATP hydrolysis. HslUV·Arc complexes were preassembled by incubating for 5 min with 10 μM ATPγS at 37 °C. Reactions in PD buffer at 37 °C were initiated by addition of ATP and a regeneration system. ( C ) HslU activation of HslV 12 (100 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM) was assayed in the presence of 100, 200, 400 or 800 nM ATPγS. ( D ) Rates of Z-Gly-Gly-Leu-AMC (200 μM) cleavage were assayed in the presence of 1 μM HslU hexamer, 100 nM ATPγS, and increasing HslV 12 . ( E ) N6-methyl-ATP supports hyperbolic activation of HslU 6 (50 nM) stimulation of HslV 12 (200 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM). The solid line is a fit (R = 0.997) to the equation activity = 100/(1+([0.85 μM][N6-methyl-ATP])).

    Journal: Journal of molecular biology

    Article Title: Asymmetric nucleotide transactions of the HslUV protease

    doi: 10.1016/j.jmb.2008.05.070

    Figure Lengend Snippet: HslU·HslV binding. ( A ) Binding of HslU (top) or E257Q HslU (bottom) to HslV 12 (15 nM) was assayed by changes in the rate of Z-Gly-Gly-AMC peptide cleavage. Reactions contained 1 mM ATPγS or 1 mM ATP and were performed in PD buffer at 25 °C. Data were fit to a quadratic form of a hyperbolic binding isotherm. Apparent K D values were 12 ± 2 nM (HslU; ATPγS), 78 ± 10 nM (HslU; ATP), 19 ± 5 nM (E257Q HslU; ATP) and 21 ± 2 nM (E257Q HslU; ATPγS). ( B (10 μM Arc-IA37-st11-titin-ssrA) strengthened binding of HslV to HslU (0.3 μM hexamer), as measured by changes in the rate of HslU ATP hydrolysis. HslUV·Arc complexes were preassembled by incubating for 5 min with 10 μM ATPγS at 37 °C. Reactions in PD buffer at 37 °C were initiated by addition of ATP and a regeneration system. ( C ) HslU activation of HslV 12 (100 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM) was assayed in the presence of 100, 200, 400 or 800 nM ATPγS. ( D ) Rates of Z-Gly-Gly-Leu-AMC (200 μM) cleavage were assayed in the presence of 1 μM HslU hexamer, 100 nM ATPγS, and increasing HslV 12 . ( E ) N6-methyl-ATP supports hyperbolic activation of HslU 6 (50 nM) stimulation of HslV 12 (200 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM). The solid line is a fit (R = 0.997) to the equation activity = 100/(1+([0.85 μM][N6-methyl-ATP])).

    Article Snippet: Triethylammonium salts of mant-ATP, mant-ATPγS, and ADP were purchased from Jena Biosciences GmbH (Jena, Germany); concentrations were determined by absorbance at 355 nm using an extinction coefficient of 5,800 M−1 cm−1 .

    Techniques: Binding Assay, Activation Assay, Activity Assay

    HslU. ( A ) Two HslU 6 ATPases (blue) can assemble with the HslV 12 peptidase (magenta). ( B ) HslU hexamers have six potential nucleotide binding sites, located at domain and subunit interfaces. From 3–6 nucleotides bind HslU 6 in different crystal structures. ( C (10 μM). Reactions components were preincubated with 10 μM ATPγS to promote HslU or HslUV association prior to addition of ATP.

    Journal: Journal of molecular biology

    Article Title: Asymmetric nucleotide transactions of the HslUV protease

    doi: 10.1016/j.jmb.2008.05.070

    Figure Lengend Snippet: HslU. ( A ) Two HslU 6 ATPases (blue) can assemble with the HslV 12 peptidase (magenta). ( B ) HslU hexamers have six potential nucleotide binding sites, located at domain and subunit interfaces. From 3–6 nucleotides bind HslU 6 in different crystal structures. ( C (10 μM). Reactions components were preincubated with 10 μM ATPγS to promote HslU or HslUV association prior to addition of ATP.

    Article Snippet: Triethylammonium salts of mant-ATP, mant-ATPγS, and ADP were purchased from Jena Biosciences GmbH (Jena, Germany); concentrations were determined by absorbance at 355 nm using an extinction coefficient of 5,800 M−1 cm−1 .

    Techniques: Binding Assay