The triple mutants [(∆clpX-lon)::cat, ∆hslVU1172::tet]

of strains L124 and Y229 were obtained through transduction with P1vir using the WE(P-) donor strain. In vivo MetA stability analysis The strains WE, L124 and Y229 were grown in M9 glucose medium at 37°C to the exponential phase (OD600 equals 0.3), treated with 200 μg/ml chloramphenicol and divided into two flasks, one of which was shifted to 44°C, while the other flask was maintained at 37°C. The samples were collected before and after chloramphenicol addition every 30 min for 2 h and prepared for Western blotting analysis as previously described [6]. Rabbit anti-MetA antibody (Peptron Inc., Daejeon, Korea) was used as the primary antibody, and horseradish peroxidase-conjugated anti-rabbit IgG antibodies (Pierce, Rockford, USA) were used as the LY2603618 price secondary antibody. The immunoblots were developed using a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, USA), scanned with a Fujifilm Image Reader LAS-3000 and analyzed with WCIF ImageJ software. Purification of MetA, measurement of enzyme activities and differential scanning calorimetry The MetA proteins were purified as described previously [11] in the presence of an EDTA-free Halt protease AZD0156 chemical structure inhibitor cocktail (Pierce, Rockford, USA). To measure the enzyme activities, the decrease in absorbance at 232 nm through the hydrolysis of the thioester bond of Selleck Apoptosis Compound Library succinyl-CoA [3] was

monitored using an ND1000 UV/Vis spectrophotometer (Nanodrop Technologies Inc., Wilmington, USA). The enzyme assays were performed in 100 mM K-phosphate buffer (pH 7.5) at 25°C for 30 min in a final volume of 20 μl. The concentrations of the substrates varied from 0.312 mM to 5 mM for L-homoserine and from 0.05 to 0.8 mM for succinyl-CoA. The reactions were initiated after the addition of 0.3 μg of native or mutant MetA. The thermal stabilities of the MetA proteins were measured calorimetrically over a temperature interval of 15-90°C at a scan rate of 90°C/h with a VP-DSC calorimeter (MicroCal, LLC, Northampton, USA) using 50 μM of protein in a 50 mM K-phosphate buffer (pH 7.5). Three scans were obtained using independent protein preparations.

In vitro MetA aggregation assay The MetA aggregates were generated after incubating 2 μM of purified protein at 45°C for 30 min, followed Sucrase by a 40-fold dilution into refolding buffer (50 mM Tris–HCl, pH 7.5, 150 mM KCl, 20 mM MgCl2 and 2 mM DTT) [33]. The soluble and insoluble protein fractions were separated through centrifugation at 14,000 g for 30 min. The soluble protein was precipitated with TCA, and the protein pellet was washed twice with ice-cold acetone, dried by speed-vac, dissolved in 20 μl of distilled water and mixed with 20 μl of 2× sample buffer. The samples (10 μl) were loaded onto a 4-15% Criterion™ TGX™ Precast Gel (Bio-Rad, Hercules, USA) and subjected to Western blotting analysis with rabbit anti-MetA antibodies.