Structure function relationships of food-related proteins/enzymes

我们NSERC资助的研究计划的长期目标是在基本的分子水平上阐明和完善食品相关蛋白质（特别是酶）的结构-功能关系（SFR）的性质。在食品领域，天冬氨酸蛋白酶（AP）作为加工助剂在多种产品的制造中起着突出的作用，例如，凝乳酶用于在干酪生产中使牛奶凝固。AP的特征在于各种属性，例如，两个催化天冬氨酸残基位于共享序列同源性的两个短氨基酸段中，在酸性pH值下具有最佳催化活性，在大的疏水性氨基酸之间具有断裂偏好，以及胃酶抑制剂的抑制作用。虽然大多数AP符合这些特征，但存在差异（例如，pH稳定性，底物特异性，糖基化），使AP的SFR研究的一个很好的模型。本研究是我们利用蛋白质工程和物理化学技术相结合对AP的SFR进行基础性、机制性研究的继续，有3个目标：1）通过研究寄生虫的SFR来评估天冬氨酸蛋白酶的SFR的普遍性，（恶性疟原虫）和植物（拟南芥）AP; 2）通过阐明因子（例如，前片段影响）使用诸如中子散射和量热法的技术对这些现象的影响;以及最后3）进一步探索合理的重新设计策略的使用（即，在开发新的工程功能性（例如，在AP前片段中包含生物活性肽序列及其对肽和AP的活性和SFR的影响）。从所提出的研究中产生的数据无疑将有助于进一步完善对AP的SFR的机制理解，并且实用地，有效地使用和设计具有所需和/或新功能的蛋白质。

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).