Engineering surface charges in a subtilisin

Abstract

Introduction of multiple charged amino acid residues in the subtilisin Savinase by genetic engineering allowed us to modify the electrostatic properties of this enzyme in a systematic way. The effects of these charge changes were investigated theoretically with the calculated electrostatic potential at the enzyme surface and experimentally using ion exchange chromatography. Our results indicate that the effect of introducing charged residues at the enzyme surface depends on the local electrostatic potential. The effects are purely additive for residues that are not too closely packed at the enzyme surface. Although it is generally accepted that polarization effects are relatively small, our data show that substantial charge shifts arise when the dominating effect of the overall charge is taken away. These shifts are not well quantified using current methods to calculate the electrostatic potential at the enzyme surface. Our work focusses on methods that will provide a better description of this surface potential.