Molecular Structural Studies of Proteins and Enzymes Important to Biotechnology

我的研究项目侧重于开发一种分子结构框架，用于理解和操纵对生物技术重要的蛋白质。我的研究项目的发现已经并将有助于吸引投资和培训加拿大生物技术的HQP。本提案中的三个主要项目遵循统一的结构生物学方法，在全球生物技术前沿的系统上取得进展。第一个项目旨在设计用于可逆偶联应用的新型链亲和素。我建议使用我们最近确定的链霉亲和素与sbp标签结合的结构来设计新一代具有商业吸引力的亲和试剂。二硫化物连接的变体和其他设计正在被表达和表征。产生最佳耦合效率和动力学的设计将被结构表征，以帮助提高结合亲和力和特异性。***第二个项目将揭示植物磷酸蛋白磷酸酶（PPP）的新型结构-功能关系。AtRLPH2的初步工作已经产生了衍射良好的晶体（dmin=2.0Å）。结构测定将使用硒代蛋氨酸或钨酸盐衍生物。具有肽配体和靶向亚基的复合物的新结构也将为理解特异性、催化和靶向的机制提供基础。***第三个项目建立了一个分子结构框架，以了解负责苯基异喹啉生物碱（BIA）生物合成的关键酶，其中包括常用的镇痛药可待因和吗啡，以及许多抗菌、止咳和抗癌化合物。我们最初的工作是从Thalictrum flavum中获得了衍射良好的PavNMT晶体，并且我们最近解决并改进了与甲基供体s -腺苷蛋氨酸（SAM）的二元配合物的结构到2.0 Å分辨率。我们目前正在结晶和解决两个三元配合物的结构（SAM和底物类似物抑制剂tetrahydropapaverine，或s -腺苷型同型半胱氨酸和底物pavine）。PavNMT的初始结构信息提示了甲基转移和底物特异性的机制，我们将利用位点定向诱变、酶动力学、晶体学和计算方法进一步探索这些机制。***我们还启动了对BIA生物合成重要的其他关键酶的结构研究。这些研究有望揭示对BIA生物合成所必需的结构上未表征的酶类的底物识别和催化的新见解。这一信息将为改进代谢工程计划提供关键工具，旨在产生具有新药理特性的改性BIA化合物。

My research program focuses on developing a molecular structural framework for understanding and manipulating proteins important for biotechnology. The discoveries made from my research program have helped and will help to attract investment and train HQP for biotechnology in Canada. The three main projects in this proposal follow a unified structural biology approach to make advances on systems at the leading edge of biotechnology worldwide.***The first project aims to engineer novel forms of streptavidin for reversible coupling applications. I propose to use our recently determined structure of streptavidin bound to the SBP-Tag to design a new generation of commercially attractive affinity-based reagents. Disulfide-linked variants and other designs are being expressed and characterized. The designs that yield the best coupling efficiencies and kinetics will be structurally characterized to help improve binding affinity and specificity.***The second project will reveal novel structure-function relationships in plant phosphoprotein phosphatases (PPP). Preliminary work on AtRLPH2 has yielded well-diffracting crystals (dmin=2.0Å). Structure determination will use selenomethionine or tungstate derivatives. Novel structures of complexes with peptide ligands and targeting subunits will also provide a basis for understanding the mechanisms underlying specificity, catalysis and targeting.***The third project establishes a molecular structural framework for understanding key enzymes responsible for the biosynthesis of benzylisoquinoline alkaloids (BIA), which include the commonly used analgesics codeine and morphine, as well as many antimicrobial, antitussive and anticancer compounds. Our initial work has yielded well-diffracting crystals of pavine N-methyltransferase (PavNMT) from Thalictrum flavum, and we have recently solved and refined the structure of a binary complex with the methyl donor S-adenosylmethionine (SAM) to 2.0 Å resolution. We are currently crystallizing and solving the structures of two ternary complexes (SAM and the substrate-analogue inhibitor tetrahydropapaverine, or S-adenosylhomocysteine and the substrate pavine). The initial structural information on PavNMT suggests mechanisms for methyl transfer and substrate specificity that we will further explore using site-directed mutagenesis, enzyme kinetics, crystallography and computational methods.***We have also initiated structural studies on other key enzymes important for BIA biosynthesis. These studies promise to reveal new insights into substrate recognition and catalysis for structurally uncharacterized classes of enzymes essential to BIA biosynthesis. This information will provide critical tools for improving metabolic engineering initiatives aimed at generating modified BIA compounds with novel pharmacological properties.