Abstract
Protein stability is a widely studied topic, there are still aspects however that need addressing. In this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VPR, using the thermostable structural homologue AQUI as a template. Four locations for proline substitutions were chosen to imitate the structure of AQUI. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPRΔC_N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. In addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.
Highlights
Protein stability is a widely studied topic, there are still aspects that need addressing
This work focused on VPRΔC, a cold active subtilisin-like serine protease from the proteinase K family[5]
VPRΔC contains three calcium binding sites and calcium binding is one of the most important structural factors contributing to the stability of the enzyme
Summary
Protein stability is a widely studied topic, there are still aspects that need addressing. The aim of this study was to enhance the stability of a kinetically stable, cold adapted subtilisin-like serine protease, VPR5 and gain more insight into the molecular basis of kinetic stability of proteins To this end a truncated version of VPR, VPRΔC11, was subjected to single point mutations incorporating the desired proline residues. The variants produced were the single proline variants VPRΔC_N3P, VPRΔC_I5P, VPRΔC_N238P, VPRΔC_T265P, the double proline variant VPRΔC_N3P/I5P, the triple proline variants VPRΔC_N3P/I5P/N238P and VPRΔC_N3P/I5P/T265P and lastly the quadruple proline variant VPRΔC_N3P/I5P/N238P/T265P The effects of these mutations on the properties of the enzyme were studied by circular dichroism (CD), differential scanning calorimetry (DSC), steady state fluorescence, acrylamide fluorescence quenching and Michaelis-Menten kinetics. Incorporating proline residues at distant parts of the protein displays synergic effects causing overall higher stability of the protein structure
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