Molecular structural studies of protein complexes for biotechnology and metabolic engineering

我的研究计划使用分子结构方法来理解和工程关键酶和生物技术，特别是代谢工程重要的结合蛋白。项目1旨在设计具有独特有用的氧化还原可转换结合特性的新型链霉亲和素。我们最近取得了突破性进展，发现了如何将二硫键引入链霉亲和素的关键环中，从而产生与生物素的氧化还原依赖性结合。用温和的还原剂破坏这些二硫键之一，使生物素的解离速率增加一千倍以上，这是第一次在生理条件下可逆地形成和分解紧密结合的生物素复合物。该突变蛋白对于某些应用是有用的，但是对于需要快速解离动力学的应用来说结合太紧。为了解决这一缺点，我们提出了一种基于结构的策略来设计和表征一套下一代突变蛋白，其在还原状态下具有更快的解离动力学，同时在氧化状态下保留极慢的解离动力学。这些突变蛋白将在生物技术中有许多应用，包括在本提案的项目2和项目3中发现蛋白质相互作用伙伴的直接应用。项目2探索植物磷蛋白磷酸酶（PPP）家族新成员的结构-功能关系。通过解决AtRLPH 2的结构和表征不寻常的底物特异性，我们最近发现了一种新的机制，以识别双重磷酸化底物，证实磷酸化蛋白质组学数据暗示RLPH 2在MAP激酶级联的调节。使用项目1的工具，我建议进一步探索AtRLPH 2如何调节MAP激酶，以及确定其他非经典植物蛋白磷酸酶，如AtSLP 1的基本结构-功能关系。项目3进一步发展对苄基异喹啉生物碱（BIA）化合物生物合成至关重要的酶的研究。我们对N-甲基转移酶（NMTs）的基础结构-功能研究已经建立了特异性和催化作用的关键分子机制。这些发现表明如何设计具有新特异性的NMT突变蛋白，为代谢工程创造新工具。我们还进行了BIA O-甲基转移酶的结构-功能研究，以及将蒂巴因转化为可待因和吗啡的途径的末端步骤中的酶。我们最近对可待因酮还原酶的结构-功能研究表明，新的蛋白质-蛋白质相互作用可能具有重要意义，对次生代谢产物的生物合成具有更广泛的影响。项目1中的工具将帮助我们探索这些蛋白质-蛋白质相互作用。我的研究计划侧重于将在加拿大快速发展的生物技术产业的短期应用，以及在生物学的基础知识的长期应用的问题。

My research program uses a molecular structural approach to understand and engineer key enzymes and binding proteins important for biotechnology, especially metabolic engineering. Project 1 aims to engineer novel forms of streptavidin with uniquely useful redox-switchable binding properties. We recently made a breakthrough, discovering how the introduction of disulphide bonds into a key loop in streptavidin creates redox-dependent binding to biotin. Breaking one of these disulphide bonds with a mild reducing agent increases the rate of dissociation for biotin by over a thousand-fold, allowing for the first time the reversible formation and breakdown of tight-binding biotin complexes under physiological conditions. This mutein is useful for certain applications but binds too tightly for applications where fast dissociation kinetics are required. To address this shortcoming, we propose a structure-based strategy to design and characterize a suite of next-generation muteins with faster dissociation kinetics in the reduced state while retaining extremely slow dissociation kinetics in the oxidized state. These muteins would have many applications in biotechnology, including immediate applications to discover protein interaction partners in projects 2 and 3 of this proposal. Project 2 explores structure-function relationships in novel members of the phosphoprotein phosphatase (PPP) family in plants. By solving the structure and characterizing the unusual substrate specificity of AtRLPH2, we recently discovered a novel mechanism to recognize dually phosphorylated substrates, corroborating phosphoproteomic data implicating RLPH2 in the regulation of MAP kinase cascades. Using tools from project 1, I propose to further explore how AtRLPH2 regulates MAP kinases, as well as to determine foundational structure-function relationships for other noncanonical plant protein phosphatases, such as AtSLP1. Project 3 further develops studies on enzymes central to the biosynthesis of benzylisoquinoline alkaloid (BIA) compounds. Our foundational structure-function studies of N-methyltransferasse (NMTs) have established key molecular mechanisms underlying specificity and catalysis. These findings indicate how to design NMT muteins with novel specificities, creating new tools for metabolic engineering. We have also undertaken structure-function studies of BIA O-methyltransferases, as well as the enzymes in the terminal steps of the pathway converting thebaine to codeine and morphine. Our recent structure-function studies of codeinone reductase suggest the possible importance of novel protein-protein interactions with wider implications on the biosynthesis of secondary metabolites. Tools from project 1 will help us explore some of these protein-protein interactions. My research program focuses on problems that will have both short-term applications in Canada's rapidly growing biotechnology industry, as well as long-term applications advancing basic knowledge in biology.