Structure function relationships of food-related proteins/enzymes

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The long-term goal of our NSERC funded research program has been to elucidate and refine the nature of structure-function relationships (SFRs) of food-related proteins (particularly enzymes) at a fundamental, molecular level. In the food area, aspartic proteinases (APs) play a prominent role as processing aids in the manufacture of a variety of products, e.g., chymosin is used to coagulate milk in the production of cheese. APs are characterized by various properties, e.g., two catalytic aspartate residues localized in two short amino acid stretches sharing sequence homology, optimal catalytic activity at acidic pH values, a scission preference between large, hydrophobic amino acids, and inhibition by pepstatin. While most APs conform to these characteristics, there are differences (e.g., pH stability, substrate specificity, glycosylation) that make APs an excellent model for SFR studies. The proposed research is a continuation of our fundamental, mechanistic examination of the SFRs of APs using a combination of protein engineering and physicochemical techniques and has 3 objectives: 1) to assess the universality of SFRs of aspartic proteinases by examining SFRs of parasitic (Plasmodium falciparum) and plant (Arabidopsis thaliana) APs; 2) to better understand protein folding/refolding as related to structural/functional stability by elucidating factors (e.g., influence of prosegment) that impact on these phenomena using techniques such as neutron scattering and calorimetry; and finally 3) to further explore the use of rational redesign strategy (i.e., the introduction of key structural elements that are not available through techniques such as site directed mutagenesis) in the development of novel, engineered functionalities (e.g., inclusion of a bioactive peptide sequence in an AP prosegment and its effect on the activity and the SFRs of the peptide and AP). The data generated from the proposed studies will undoubtedly aid in the further refinement of a mechanistic understanding of the SFRs of APs, and pragmatically, in the efficient use and design of proteins with desired and/or novel function(s).