L stability. The get 548-04-9 protein structures were solvated ?with Monte Carlo simulated TIP3P [42] water model with a 10 A buffer space from the protein edges in an orthorhombic box and the system was then neutralized by replacing water molecules with sodium and chloride counter ions. Similarly, unbound HtrA2 system was also developed as a control. Neutralization of systems was done by adding 2 Na+ ions in unbound HtrA2 and 4 Na+ ions for peptide bound complexes. The particle-mesh Ewald method (PME) [43] was used to calculate long-range electrostatic ?interactions with a grid spacing of 0.8 A. Van der Waals andAllosteric Regulation of HtrApurity. The fractions with .95 purity were stored in aliquots at 280uC until use.FITC-b-Casein Cleavage AssayThe proteolytic activity of wild type and the mutants were determined using FITC-labelled b-casein cleavage assay [47]. Fluorescent substrate cleavage was determined by incubating 200 nM of enzymes with increasing concentration (0?5 mM) of bcasein at 37uC in cleavage buffer (20 mM Na2HPO4/NaH2PO4, pH 8.0, 100 mM NaCl, 0.1 mM DTT). Fluorescence was monitored in a multi-well plate reader (Berthold Technologies) using excitation wavelength of 485 nm and emission at 545 nm. Reaction rates v0 (mM/min) were determined by linear regression analysis corresponding to the maximum reaction rates for individual assay condition. Assays are representative of at least three independent experiments done in triplicate. The steady-state kinetic parameters were obtained from the reaction rates by fitting data to Michaelis-Menten equation using nonlinear least squares subroutine in KaleidaGraph program (Synergy software).Supporting InformationFigure S1 Interaction of peptides with HtrA2. a. Ligplotdefined for PDZ peptide groove binding substrate. The peptide was better in terms of solubility as compared to other activating peptides and binding studies were done using Isothermal titration calorimetry. The titrations were carried out using Micro Cal ITC200 (GE Healthcare) 22948146 with the calorimetry cell containing 200 ml of wild type or N216A/S219A mutant HtrA2 in 20 mM Na2HPO4/NaH2PO4 buffer, 100 mM NaCl, pH 7.8. The concentration of protein was in range from 20 to 50 mM and was titrated with 1.5 ml injections of a solution containing 0.4 mM activator peptide reconstituted in the same buffer. To correct the effect of heat of dilution, a blank injection was made under identical conditions. All experiments were performed at 25uC and the data was analyzed using the manufacture provided MicroCal software with the integrated heat peaks fitted to a one site-binding model. Simulated ITC raw data for the protein with the activating peptide is represented in the upper panel and the integrated data in the lower panel. The dissociation constant was calculated to be 7.5 mM for wild type (left panel) and no significant heat change was observed for the SBP double mutant (right panel). (TIF)Table S1 Docking analysis of replica fragments with HtrA2. The fragments have been arranged according to their docking scores. (DOC) Table S2 Designed peptide fragments. Fragments of peptide combinations generated based on functional group studies have been enlisted. (DOC) Movie S1 Orientation of active site triad and oxyanionfor GSAWFSF with HtrA2 which BIBS39 supplier represents residues involved and the nature of interactions. b. Ligplot for GQYYFV interaction pattern with HtrA2. c. Ligplot for GPFPIIV with HtrA2 which represents residues involved and the nature of intera.L stability. The protein structures were solvated ?with Monte Carlo simulated TIP3P [42] water model with a 10 A buffer space from the protein edges in an orthorhombic box and the system was then neutralized by replacing water molecules with sodium and chloride counter ions. Similarly, unbound HtrA2 system was also developed as a control. Neutralization of systems was done by adding 2 Na+ ions in unbound HtrA2 and 4 Na+ ions for peptide bound complexes. The particle-mesh Ewald method (PME) [43] was used to calculate long-range electrostatic ?interactions with a grid spacing of 0.8 A. Van der Waals andAllosteric Regulation of HtrApurity. The fractions with .95 purity were stored in aliquots at 280uC until use.FITC-b-Casein Cleavage AssayThe proteolytic activity of wild type and the mutants were determined using FITC-labelled b-casein cleavage assay [47]. Fluorescent substrate cleavage was determined by incubating 200 nM of enzymes with increasing concentration (0?5 mM) of bcasein at 37uC in cleavage buffer (20 mM Na2HPO4/NaH2PO4, pH 8.0, 100 mM NaCl, 0.1 mM DTT). Fluorescence was monitored in a multi-well plate reader (Berthold Technologies) using excitation wavelength of 485 nm and emission at 545 nm. Reaction rates v0 (mM/min) were determined by linear regression analysis corresponding to the maximum reaction rates for individual assay condition. Assays are representative of at least three independent experiments done in triplicate. The steady-state kinetic parameters were obtained from the reaction rates by fitting data to Michaelis-Menten equation using nonlinear least squares subroutine in KaleidaGraph program (Synergy software).Supporting InformationFigure S1 Interaction of peptides with HtrA2. a. Ligplotdefined for PDZ peptide groove binding substrate. The peptide was better in terms of solubility as compared to other activating peptides and binding studies were done using Isothermal titration calorimetry. The titrations were carried out using Micro Cal ITC200 (GE Healthcare) 22948146 with the calorimetry cell containing 200 ml of wild type or N216A/S219A mutant HtrA2 in 20 mM Na2HPO4/NaH2PO4 buffer, 100 mM NaCl, pH 7.8. The concentration of protein was in range from 20 to 50 mM and was titrated with 1.5 ml injections of a solution containing 0.4 mM activator peptide reconstituted in the same buffer. To correct the effect of heat of dilution, a blank injection was made under identical conditions. All experiments were performed at 25uC and the data was analyzed using the manufacture provided MicroCal software with the integrated heat peaks fitted to a one site-binding model. Simulated ITC raw data for the protein with the activating peptide is represented in the upper panel and the integrated data in the lower panel. The dissociation constant was calculated to be 7.5 mM for wild type (left panel) and no significant heat change was observed for the SBP double mutant (right panel). (TIF)Table S1 Docking analysis of replica fragments with HtrA2. The fragments have been arranged according to their docking scores. (DOC) Table S2 Designed peptide fragments. Fragments of peptide combinations generated based on functional group studies have been enlisted. (DOC) Movie S1 Orientation of active site triad and oxyanionfor GSAWFSF with HtrA2 which represents residues involved and the nature of interactions. b. Ligplot for GQYYFV interaction pattern with HtrA2. c. Ligplot for GPFPIIV with HtrA2 which represents residues involved and the nature of intera.
