Spore photoproduct lyase (SPL) catalyzes the repair of the UV lesion

Spore photoproduct lyase (SPL) catalyzes the repair of the UV lesion spore photoproduct (SP) in a reaction dependent on S-adenosyl-L-methionine (SAM). binding. was expressed using Tuner(DE3)-pLysS cells transformed with a pET14b expression vector made up of the gene. The resulting protein was produced in minimal media and purified anaerobically by Ni-HisTrap chromatography and FPLC as previously described [29 30 The protein was anaerobically dialyzed in 20 mM sodium phosphate 350 mM NaCl 5 glycerol pH 7.5. The protein was then concentrated using an Amicon concentrator fitted with an YM-10 membrane to a final concentration of ~650 μM. All protein samples used in assays were prepared in the MBRAUN box (O2 ≤ 1 ppm) unless pointed out otherwise. The protein and iron concentration were determined by methods previously described [31 32 SAM was synthesized as previously described [33]. 2.2 Preparation of enzyme/ligand mixtures A 6-mer oligonucleotide (5′-GCAAGT-3′ and complement 5′-ACT TGC-3′) were obtained from Integrated DNA Technologies (Coralville IA). Equimolar amounts of each strand were mixed in water and then annealed by heating to boiling followed by removal of the heat source and slow cooling of the water heat to 25 °C. Proteins were prepared in advance in a buffer consisting of 40 mM sodium phosphate 350 mM NaCl 5 glycerol pH 7.5 under appropriate anaerobic conditions. Protein solutions were diluted to an identical concentration (250 μM final concentration) for all those assays. A ligand answer or control buffer was added to each of the matched control/experimental protein samples. The ligand solutions and their final concentrations were: 6mer oligo (1.0 or 2.0 mM) synthetic 5= 0.01 min) the solvent composition was changed via the binary pump to 100% “B”: the residual water in the system and column were sufficient to slightly delay the elution of the protein from the flow-through. The PF-3845 protein elution peak was centered at approximately 0.4 min. Following protein elution at 1.21 min the solvent composition was changed back to 20% B for column re-equilibration. The mass spectra were obtained on a Bruker micrO-TOF Mass Spectrometer equipped with an ESI source. The capillary exit voltage was 120 V and gas heat was 200 °C. All data were recorded in positive mode between 300 and 3000 in profile mode. The hardware summation time was 1 s with no rolling averaging. Quenching of the H/D exchange reaction was a consequence of sample injection into the HPLC system. A flow rate of 600 μL/min carried the sample from the autosampler (maintained at 25 °C) to the column compartment and reverse-phase column (maintained at 4 °C) in less than 2 s. Although this heat produces more back-exchange compared to 0 °C it is more stable. The solvents also provide quenching due to the low pH and reduced water composition of the loading solvent: 80/20 H2O/acetonitrile with ILKAP antibody 0.1% (v/v) formic acid. A control reaction was run immediately following each experimental reaction to account for hidden variation in the instrumentation and replicates of control/experiment reaction pairs were conducted on individual days to ensure unbiased results. 2.5 Analysis of H/D exchange (HDX) data With the described chromatographic system the protein eluted at 0.4 min during the 2 min run. The resulting mass spectra were PF-3845 then averaged and processed using the Data Analysis 4.0 software suite supplied by Bruker Daltonics. The Maximum Entropy routine was used for charge-state deconvolution of the natural data which were then exported into PF-3845 text files: these actions were automated via scripts to ensure reproducibility. The deconvoluted spectra were processed using Python and Scipy scripts and a reference spectrum PF-3845 of a 0% D2O sample was used to calculate the cross-correlation with the experimental spectra for the true HDX reactions. The cross-correlation function produced a symmetric peak centered at the deuterium uptake of the experimental sample describing the shift between reference and experimental peaks. These calculations were manually verified against the more common centroid peak assignment method but the use of cross-correlation was less sensitive to bias permitting use of a fully-automated processing workflow. The shape and symmetry of the cross-correlation shift plot also provided.