Properties of SLPI, a protease inhibitor from Streptomyces lividans and its binding properties to subtilases

The goal of this research project was to use isothermal titration calorimetry (ITC) to characterize the binding of a protease inhibitor to the structural homologues VPR, from a psychotropic Vibrio species, and AQUI, from the thermophile Thermus aquaticus. ITC is a powerful tool and with a single binding isotherm several thermodynamic binding parameters can be determined, including reaction stoichiometry, dissociation constant (Kd) and hence binding free energy (ΔG), enthalpy (ΔH) and entropy (ΔS). The objective was to determine if these parameters would differ, reflecting different thermodynamic driving forces of binding to these two protease, which differ greatly with respect to temperature adaptation. In order to use ITC to determine these parameters a suitable inhibitor was needed. Many members of the Streptomyces family are known for secreting protease inhibitors belonging to the Streptomyces subtilisin inhibitor family of inhibitors. DNA from Streptomyces lividans TK24 was acquired and from it the sequence for Streptomyces lividans protease inhibitor (SLPI) was cloned into E. coli and overexpressed. SLPI was characterized using circular dichroism (CD) spectoscopy, fluorescence spectroscopy, differential scanning calorimetry (DSC) and ITC. SLPI was determined to be a thermodynamically stable homodimer capable of refolding after thermal denaturation. DSC revealed a non-two-state denaturation process, with indication of cooperative unfolding of SLPI and its dimer interface. Guanidinium chloride (GdmCl) was used in an attempt to separate the two events but proved unsuccessful. Since denaturation curves, observed either with fluorescence or CD, showed only a single cooperative transition between native and denatured states. Thermal stability of SLPI was thoroughly characterized with DSC, with several constants, such as ΔH and ΔG of unfolding and heat capacity (ΔCp) determined. Complexes of SLPI with AQUI and VPR were also characterized with no major structural changes detected by CD and fluorescence. DSC thermograms of the complexes revealed an exothermic peak, overlapping with the endothermic denaturation peaks, which was dependent on SLPI being present in excess of the protease. The cause of this exothermic peak could not be established, with further studies needed to determine its exact nature. Finally, binding of SLPI to VPR and AQUI was characterized with ITC measurements. The measurements proved rather irreproducible, likely as a result of SLPI having a Kd value below 10-9 M for both VPR and AQUI. From what could be gathered, there is no significant difference between the binding of SLPI to VPR or AQUI. This runs counter to the theory that psychrophilic enzymes acquire their cold activity through an increase in flexibility in the active site. As a more flexible active site should result in a difference in binding enthalpy