Using Structural Studies to Investigate the Relationships Between Enzymes and Ligands.

我的研究重点是利用蛋白质结构来理解生物系统中分子如何相互识别。我的实验室通过使用结构研究来加强对酶及其底物之间的相互作用的理解，从而建立了自己的声誉。这使我们能够预测相互作用中的变化将对酶产生的影响。结晶学与酶学、计算研究和其他生物物理技术相结合，我们可以表征配体与蛋白质相互作用的方式。我们的长期目标是利用这些信息开发能够影响酶过程的化合物，并了解酶系统的进化。我们正在使用硫氧还蛋白系统作为研究蛋白质-蛋白质相互作用的模型系统。硫氧还蛋白系统是氧化应激中的关键系统。我们的研究加深了我们对硫氧还蛋白(Trx)和硫氧还蛋白还原酶(TrxR)之间相互作用的理解。我们预测，我们可以设计出对TrxR或Trx具有特异性的多肽。结构研究、酶学和其他监测蛋白质-多肽结合的技术(如ITC和SPR)将有助于改善多肽结合。了解酶结构-功能关系的进化仍然是我的实验室的一个关键重点。我们将继续使用硫氧还蛋白系统来继续研究Trx和TrxR在极端条件下的相互作用方式。我们之所以选择这个系统，是因为它的化学特性非常好，TRX系统无处不在，而且氧化应激具有很大的科学价值。利用结晶学和酶学，我们将进一步表征极端细菌硫氧还蛋白体系之间的相互作用。充分了解决定TrxR/Trx复合体识别和结合的相互作用将为深入了解其他蛋白质-蛋白质相互作用提供依据。我们的工作还包括了解小分子是如何被酶识别的，以及这种结合如何影响酶的行为。二氢二氢吡啶甲酸合成酶(DHDPS)是由变构位点的赖氨酸调节的。我们测定了DHDPS在底物和抑制剂存在和不存在的情况下的结构，以研究该酶的变构抑制是如何发生的。我们的假设是，抑制剂诱导的灵活性变化是活性丧失的原因。我们将利用生物信息学、酶学和分子动力学研究来靶向特定的氨基酸，以探索控制变构抑制的柔性/刚性区域。这些研究将极大地增加我们对灵活性变化如何影响酶行为的了解。我们拥有最先进的设施，包括位于萨斯喀彻温大学的CVS、SSSC和PCCF，使我们能够在研究蛋白质结构-功能关系方面处于领先地位，在接下来的五年里，我们将继续研究新的系统，以解决关于蛋白质的重要问题：配体相互作用。

My research is focused on using protein structures to understand how molecules recognize each other in biological systems. My lab has established its reputation by using structural studies to enhance understanding of the interactions between enzymes and their substrates. This allows us to predict the effects that changes in the interactions will have on enzymes. Combining crystallography with enzymology, computational studies, and other biophysical techniques, we can characterize the ways that ligands interact with proteins. We have the long-term goal of using this information to develop compounds that can affect enzyme processes and to understand the evolution of enzyme systems. We are using the thioredoxin system as a model system for studying protein-protein interactions. The thioredoxin system is a critical system in oxidative stress. Our studies have increased our understanding of the interactions between thioredoxin (Trx) and thioredoxin reductase (TrxR). We predict that we can design peptides that exhibit specificity towards TrxR or Trx. Structural studies, enzymology, and other techniques to monitor protein-peptide binding (eg ITC and SPR) will help improve peptide binding. Understanding the evolution of enzyme structure-function relationships remains a key focus of my lab. We will continue to use the thioredoxin system to continue to study the way Trx and TrxR interact under extreme conditions. We have chosen this system because the chemistry is very well characterized, the Trx system is ubiquitous and oxidative stress is of great scientific interest. Using crystallography and enzymology we will further characterize the interactions of the thioredoxin system of extremophiles. A full understanding of the interactions that determine the recognition and binding of the TrxR/Trx complex will offer insights into other protein-protein interactions. Our work also involves understanding how small molecules are recognized by enzymes and how this binding affects enzyme behavior. Dihydrodipicolinate synthase(DHDPS) is regulated by lysine at an allosteric site. We have determined structures of DHDPS in the presence and absence of substrates and inhibitors, to study how the allosteric inhibition of this enzyme occurs. Our hypothesis is that inhibitor-induced changes in flexibility are responsible for the loss in activity. We will use bioinformatics, enzymology, and molecular dynamic studies to target specific amino acids to probe the flexible/rigid regions in controlling allosteric inhibition. These studies will greatly increase our knowledge in how flexibility changes affect the behavior of enzymes. Our access to state-of-the-art facilities, including the CVS, the SSSC, and the PCCF located at the University of Saskatchewan enables us to be leaders in studying protein structure-function relationships and over the next five years we will continue to investigate novel systems to address important questions about protein:ligand interactions.