mant atpγs  (Jena Bioscience)

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: ( 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. ). 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 , but with PAN-R328/331A (1 μM). Quantifications are presented on . ( 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: Mutagenesis, Variant Assay, Binding Assay

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: ( 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: Fluorescence, Binding Assay, Negative Control, Concentration Assay

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: ( 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: