Sandbox Reserved 1546: Difference between revisions

<p>• A plasmid encoding the His-tagged catalytic domain of human Gcn5L2 under T7 induction was obtained and the protein was expressed and purified <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. The purified protein was then dialyzed into 20MM and concentrated to 9 mh ml- flash-frozen in liquid nitrogen and stored at -80°C <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• Kinetic measurements were used to compare rates of acetylation propionylation and butrylation using a DNTB assay with a few modifications <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• The reaction was then incubated for five minutes a 37°C for the remainder of the experiment before adding acyl-CoA and was maintained at that temperature for the remainder of the experiment <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. </p>

<p>• Six data points were collected to find a time frame over which acyl-CoA consumption was linear over time <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. The reaction was then quenched at the indicated time points by the addition of two volumes of quenching buffer <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• After all the samples were collected, one column of 4mM DTNB was dissolved in 100mM sodium phosphate at pH 6.8 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Samples were then moved to a 384-well polystyrene clear-bottom plate and the absorbance at 412 nM was measured in a POLARstar Omega plate reader <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. The absorbances were converted to concentrations using a standard curve generated by reacting increasing concentrations of CoA with DTNB using an extinction coefficient for 3-thio-6-nitrobenzoate (TNB) of 412 nm = 13,700 M−1 cm−1 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Subsequent reactions were performed in triplicate and quenched after 0.5 minutes (acetyl-CoA), 5 minutes (propionyl-CoA) or 20 minutes (butyryl-CoA) <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• All acylation rates were corrected by subtracting the rate of acyl-CoA consumption by Gcn5L2 in the absence of a peptide <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Steady-state kinetic titrations varying the acetyl-CoA or butyryl-CoA concentration were then performed with a continuous spectrophotometric assay as previously described <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• The acetyl-CoA or butyryl-CoA concentration was varied briefly between 0.25 and 100 µM in the presence of 50 nM hsGcn5L2 and 300 µM histone H3 peptide <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Reactions were performed in a total volume of 50 µl at 37°C in 384-well plates (Greiner Bio-One) and were initiated with the acyl-CoA <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. The absorbance at 340 nm was monitored continuously using a POLARstar Omega plate reader (BMG Labtech) for 5–20 min and converted into the molar concentration of NADH using Beer's law, assuming ∊340 nm = 6220 M−1 cm−1 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. For controlled variables, rate measurements were performed at each concentration of acyl-CoA in the absence of peptide <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Each measurement was performed in triplicate, and reaction velocities in the presence of peptide were blanked by the rate of reaction in the absence of peptide <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• Blanked rates were normalized to enzyme concentration, plotted as a function of substrate concentration, and fitted to the Michaelis–Menten equation using nonlinear least-squares regression in GraphPad Prism 5 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Butyryl-CoA inhibition measurements were also performed with the enzyme-coupled assay <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Reaction velocities were measured in the presence of 0.5–10 µM acetyl-CoA and 50 nM hsGcn5L2 with increasing concentrations of butyryl-CoA (0, 50, 100 or 300 µM) <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Under these conditions, the consumption of butyryl-CoA by hsGcn5L2 was undetectable by the same assay <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Blanked rates were normalized to enzyme concentration and the resulting curves were globally fitted to a competitive-inhibition model in GraphPad Prism 5 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• Propionyl-CoA and butyryl-CoA were diluted in 20 mM HEPES pH 7.5 and stored at −20°C at a concentration of 20 mM as calculated using an absorbance of 260 nm = 16,400 M−1 cm−1 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Purified human Gcn5L2 (amino acids 497–662) were mixed with each acyl-CoA to produce a final concentration of 1.6 mM acyl-CoA and 7.9 mg ml−1 protein <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. NaCl was added for a final concentration of 125 mM from a 5 M stock, and the resulting mixture was incubated on ice for 30 minutes <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Both complexes were crystallized using hanging-drop vapor diffusion by mixing 1 µl protein–acyl-CoA complex solution with 1 µl well solution <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Human Gcn5L2 bound to propionyl-CoA was crystallized in 20%(v/v) ethanol, 100 mM Tris pH 9.0. Human Gcn5L2 bound to butyryl-CoA was crystallized in 10%(v/v) 2-propanol, 3% glycerol, 100 mM HEPES pH 7.8. <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/></p>

<p>• Crystals were then cryoprotected by soaking in a well solution supplemented with 9% sucrose, 4% glucose, 8% ethylene glycol and 8% glycerol <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Prior to data collection, crystals were flash-cooled in a liquid-nitrogen stream <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• This step composed of data collection and analysis from the experiment <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• This step composed of data entry into the Protein Data Bank (PDB) <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>.</p>

<p>• Gcn5 is a weak acetyltransferase <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/></p>

<p>• Discovery of the structures of a Hcn5L2 bound to propionyl-CoA and butyryl-CoA <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/> </p>

<p>• Human Gcn5L2 efficiently acetylates and propionylates peptides, while its butyrylating activity is nearly undetectable <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/></p>

<p>• Construction of a model of the ternary complex with peptide and CoA <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/></p>

<p>• Butyryl-CoA is a competitive inhibitor of acetylation by human Gcn5 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/></p>

<p>• Gcn5L2 propionylates histone peptides approximately ninefold more slowly and butyrylates peptides nearly 400-fold more slowly compared with its acetyltransferase activity <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/></p>

<p>• Based on these relative rate measurements, Gcn5L2 is unlikely to contribute significantly to lysine butyrylation in vivo but may be capable of catalyzing lysine propionylation under physiological conditions <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/></p>

The findings of the experiment helps future researchers to be able to determine crystal structures that are able to describe how Gcn5 accommodates propionyl-CoA in the active site <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. The results of the experiment have also provided a structural mechanism that explains how the Gcn5 discriminates between different acyl-CoA molecules <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. Further data indicate that butyryl-CoA is more of a competitive inhibitor than acetyl-CoA for human Gcn5 <ref name = "Structural basis for acyl-group discrimination by human Gcn5L2"/>. This then raises the question as to whether fluctuating the levels of acyl-CoA molecules in cells may regulate the activity of Gcn5, which may possibly be tested in future experimentations.