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

智力MeritBeta碳酸酐酶（beta-CA）是利用碳酸氢盐的酶，对于许多细菌和酵母的最佳生长至关重要。许多细菌β-CA具有独特的非催化碳酸氢盐结合位点，其被认为通过构象变化来调节酶，所述构象变化需要多链蛋白质复合物中的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没有很好的理解，尽管事实上变构酶在所有生物体的代谢调节中既常见又重要。该研究项目将使用分子生物学（定点诱变），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）的方法来探测β-CA亚基间通讯的分子细节，并严格测试潜在作用机制的假设。β-CA亚基间通讯的分子机制的知识不仅提供了深入了解这一特定的（并建议可能的手段中断其在宿主细菌中的适当功能），但也将提供基础知识的应用程序的变构酶的亚基间通讯的机制。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先进的，当前的实践培训。这些学生中有很大一部分在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队伍。此外，一名对本科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。书面实验室指南适合于涉及本科生或其他新手研究人员在蛋白质工程和X射线晶体学的现代方法将更新和免费传播给科学界使用公开访问的维基。最后，收购关键实验室自动化将大大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.