Research objectives and content
In this project we propose to study the pH dependent electrostatic contribution to the pH-activity profile of a model enzyme, Fusarium solani pisi cutinase, as well as charge mutants thereof, with the goal of understanding in more detail one of the most important effects contributing to the pH-dependency of enzyme activity. We will use rational Protein Engineering to introduce, remove or move charges that we believe contribute to the pH activity profile. In all three cases we will substitute one residue in the native chain with another, and the residue to be changed will either be the charged residue or the neighboring residue. By modifying the latter we can move the charge without changing the nature of the charged residue. For all mutants we will measure the pH-activity profile using a standard lipolytic assay. We will also investigate the activity changes that accompany the changes in the media composition and correlate the observations to shifts in the electrostatic environment, that will be mapped on the molecular surface each protein (native and mutants). The 3D structures of the mutants will be solved in an X-ray laboratory collaborating with the host institution. ITC measurements will allow us to do thermodynamic studies of cutinase interactions (both native and mutated cutinase) with its substrats and inhibitors. DSC will be used in order to study structural transitions in native and mutated cutinase, as a function of time and temperature, in the absence and presence of substrat and inhibitors. Both measurements will be obtained under different media composition, where the electrostatic properties changed These studies will give us valuable novel insight into protein structure-function relationship.
Training content (objective, benefit and expected impact)
The overall aim of the present project is to develop better models for the description of electrostatic effects on protein activity, namely on the pH-activity profile of the enzyme. This meets the goals of the biotechnological and pharmaceutical science and industry to establish an atomic understanding of protein function. Since cutinase is an enzyme which is secreted by phytopathogenic fungi and bacteria in order to invade and infect plants, we envision that such knowledge could be of direct significance for the design and composition of anti-fungal and bacterial agents, and in a broader sense lead to generic knowledge broadly applicable to a host of relevant protein structure function questions central to the European industry and science.
Links with industry / industrial relevance (22)
Cooperating Partners in Related Projects:
UNILEVER, PHARMACIA, EUROPEAN GENENCOR