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提炼与食品相关的蛋白质(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)在各种产品的生产中扮演着重要的加工助剂的角色，例如在奶酪生产中，凝乳酶被用来凝固牛奶。AP具有多种性质，例如，位于两个氨基酸短链上的两个催化天冬氨酸残基具有相同的序列同源性，在酸性pH值下具有最佳的催化活性，在大的疏水氨基酸之间具有断裂偏好，以及被胃抑素抑制。虽然大多数AP符合这些特征，但也有一些差异(例如，pH稳定性、底物专一性、糖基化)使AP成为SFR研究的优秀模型。这项拟议的研究是我们使用蛋白质工程和物理化学技术相结合对AP的SFRS的基础和机制的继续，并有3个目标：1)通过检测寄生的(恶性疟原虫)和植物(拟南芥)AP的SFRS来评估天冬氨酸蛋白酶SFRS的普遍性；2)通过利用中子散射和量热等技术阐明影响这些现象的因素(例如前段的影响)，以更好地了解蛋白质折叠/重折叠与结构/功能稳定性的关系；最后，3)进一步探索合理的重新设计策略(即，引入通过定点突变等技术无法获得的关键结构元件)在开发新颖的工程功能(例如，在AP前段中包括生物活性多肽序列及其对多肽和AP的SFRs的影响)中的使用。从拟议的研究中产生的数据无疑将有助于进一步完善对AP的SFR的机制的理解，并在实用上有助于有效地使用和设计具有所需和/或新功能的蛋白质(S)。

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).