RUI: Structural Biology and Mechanism of Intersubunit (Allosteric) Communication in beta-Carbonic Anhydrase

智力优点贝塔碳酸酐酶(β-CA)是利用碳酸氢盐的酶，对于许多细菌和酵母的最佳生长是必不可少的。许多细菌β-CA具有独特的非催化碳酸氢盐结合部位，被认为是通过构象变化来调节酶的，这种构象变化需要多链蛋白质复合体中各个蛋白质链之间的协调通信。这种链间(变构)通讯的分子基础对于这种和许多其他变构酶还不是很清楚，尽管变构酶在所有生物体的新陈代谢调节中都是常见的和重要的。本研究项目将使用分子生物学(定点突变)、生物物理化学(酶动力学和可见光谱)和结构生物学(X射线衍射)的方法来探索β-CA亚单位间通讯的分子细节，并严格检验潜在作用机制的假说。了解β-CA亚单位间通讯的分子机制不仅有助于深入了解这一特殊情况(并提出可能的方法来阻断其在宿主细菌中的正常功能)，而且还将为变构酶亚单位间通讯机制的应用提供基础知识。更广泛的影响研究项目有几个更广泛的影响，其中之一是本科生参与研究项目，提供蛋白质化学和结构生物学方面的高级当前实践培训。这些学生中有很高比例在PI的实验室接受培训，进入物理和生命科学的高级学位课程，并最终加入国内科学工作大军。此外，对本科生研究和教育教学感兴趣的博士后研究员将接受生物物理化学和结构生物学方面的培训。将更新适合让本科生或其他新手研究人员参与蛋白质工程和X射线结晶学现代方法的书面实验室指南，并使用可公开访问的维基向科学界免费传播。最后，收购关键的实验室自动化将显著提高高露洁大学生物物理化学研究基础设施的效率和生产力。

Intellectual MeritBeta carbonic anhydrases (beta-CAs) are bicarbonate-utilizing enzymes that are essential for optimal growth in many bacteria and yeast. Many bacterial beta-CAs have a unique non-catalytic bicarbonate binding site that is thought to regulate the enzyme via a conformational change that requires coordinated communication between individual protein chains in a multi-chain protein complex. The molecular basis of such inter-chain (allosteric) communication is not well-understood for this and many other allosteric enzymes, despite the fact that allosteric enzymes are both common and important in the regulation of metabolism in all organisms. This research project will use methods of molecular biology (site-directed mutagenesis), biophysical chemistry (enzyme kinetics and visible spectroscopy) and structural biology (X-ray diffraction) to probe the molecular details of intersubunit communication in beta-CA and critically test hypotheses of potential mechanisms of action. Knowledge of the molecular mechanism of intersubunit communication in beta-CA will not only provide insight into this particular (and suggest possible means of interrupting its proper function in host bacteria) but will also provide fundamental knowledge for application to mechanisms of intersubunit communication for allosteric enzymes. Broader impactsThere are several broader impacts of the research project, one of which is the involvement of undergraduate students in research projects that provide training in advanced, current praxis in protein chemistry and structural biology. A high percentage of these students, trained in the PI's lab, enter into advanced degree programs in the physical and life sciences, and ultimately join the domestic scientific work force. In addition, a post-doctoral fellow with an interest in instruction in undergraduate research and education will be trained in biophysical chemistry and structural biology. Written laboratory guides appropriate for involving undergraduates or other novice investigators in modern methodologies of protein engineering and X-ray crystallography will be updated and freely disseminated to the scientific community using a publicly accessible wiki. Finally, the acquisition of critical laboratory automation will significantly enhance the efficiency and productivity of the biophysical chemistry research infrastructure at Colgate University.