Protein engineering the surface of enzymes

The protein surface is the interface through which a protein senses the ext ernal world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A tota l of 95% of all titratable residues are located on the surface of soluble p roteins. In evolutionary related families of proteins such residues are par ticularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% expo sed on the surface and the remainder were < 30% exposed. The individual res idue types show distinct differences. The data presented provides an import ant new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cu tinase family of enzymes has been investigated. The stability of native cut inase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activi ty is linked with the deprotonation of the active site HIS188, destabilizat ion of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling in vestigation of the pH dependence of the electrostatic potentials reveals th at the activity range is accompanied by the development of a highly signifi cant negative potential in the active site cleft. The 3D structures of thre e mutants of the Fusarium solani pisi cutinase have been solved to high res olution using X-ray diffraction analysis. Preliminary X-ray data are presen ted. (C) 1998 Published by Elsevier Science B.V. All rights reserved.