Molecular Structural Studies of Proteins and Enzymes Important to Biotechnology

我的研究计划侧重于开发分子结构框架，以理解和操纵对生物技术重要的蛋白质。从我的研究计划中所取得的发现已经并将有助于吸引投资和培养加拿大生物技术的HQP。该提案中的三个主要项目遵循统一的结构生物学方法，以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霉亲和素。我建议使用我们最近确定的结构链霉亲和素绑定到SBP标签设计新一代的商业吸引力的亲和试剂。二硫键连接的变体和其他设计正在表达和表征。产生最佳偶联效率和动力学的设计将在结构上表征以帮助改善结合亲和力和特异性。 第二个项目将揭示植物磷蛋白磷酸酶（PPP）的新结构-功能关系。对AtRLPH 2的初步研究已经产生了衍射良好的晶体（dmin= 2.0 nm）。结构测定将使用硒代甲硫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、催化和靶向的潜在机制提供基础。 第三个项目建立了一个分子结构框架，以了解负责苄基异喹啉生物碱（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。我们的初步工作已经产生了衍射晶体的番石榴N-甲基转移酶（PavNMT）从Thatritrum flavum，我们最近已经解决和完善的结构的二元复合物与甲基供体S-腺苷甲硫氨酸（SAM）到2.0 μ m分辨率。我们目前正在结晶和解决两个三元复合物（SAM和底物类似物抑制剂四氢罂粟碱，或S-腺苷高半胱氨酸和底物pavine）的结构。PavNMT的初始结构信息表明甲基转移和底物特异性的机制，我们将进一步探讨使用定点诱变，酶动力学，晶体学和计算方法。 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。这些信息将提供关键的工具，以改善代谢工程的倡议，旨在产生新的药理学特性的改性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.