Master's lecture in Biochemistry - Sveinn Bjarnason

Master's student: Sveinn Bjarnason

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

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Faculty: Faculty of Physical Sciences

Advisors: Magnús Már Kristjánsson and Kristinn R. Óskarsson

Examiner: Ólafur Þór Magnússon

Abstract

The goal of this research project was to characterize the binding of a selected protease inhibitor to the structural homologues VPR and AQUI, which are subtiliisin-like proteases from a psychotropic Vibrio species and the thermophile Thermus aquaticus, respectivly. Several thermodynamic binding parameters were determined, including reaction stoichiometry, dissociation constant (Kd) and binding free energy (ΔG), enthalpy (ΔH) and entropy (ΔS). The objective was to determine if these parameters differed reflecting different thermodynamic driving forces of binding to these two proteases, differing greatly with respect to temperature adaptation. In order to determine these parameters a suitable inhibitor was needed. Many members of the Streptomyces family are known to secrete protease inhibitors belonging to the socalled Streptomyces subtilisin inhibitor family. Thus, DNA from Streptomyces lividans TK24 was acquired and from it the gene for Streptomyces lividans protease inhibitor (SLPI) was cloned into E. coli and overexpressed. SLPI was characterized using circular dichroism spectoscopy (CD), 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. 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. Finally, binding of SLPI to VPR and AQUI as characterized with ITC measurements will be reported.