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

碳酸酐酶（β - cas）是一种利用碳酸氢盐的酶，对许多细菌和酵母的最佳生长至关重要。许多细菌β - cas具有独特的非催化性碳酸氢盐结合位点，该位点被认为通过需要在多链蛋白质复合物中单个蛋白质链之间协调通信的构象变化来调节酶。尽管变构酶在所有生物体的代谢调节中既常见又重要，但这种链间（变构）通信的分子基础对这种变构酶和许多其他变构酶还没有很好地理解。该研究项目将使用分子生物学（位点定向诱变）、生物物理化学（酶动力学和可见光谱学）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，并严格检验潜在作用机制的假设。了解β - ca亚基间通讯的分子机制不仅有助于深入了解这一特殊情况（并提出在宿主细菌中中断其正常功能的可能方法），还将为应用于变构酶亚基间通讯机制提供基础知识。更广泛的影响该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，为蛋白质化学和结构生物学的先进、最新实践提供培训。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，并最终加入国内的科学工作队伍。此外，有兴趣指导本科生研究和教育的博士后将接受生物物理化学和结构生物学方面的培训。适合本科生或其他新手研究蛋白质工程和x射线晶体学的现代方法的书面实验室指南将被更新，并使用可公开访问的维基自由传播到科学界。最后，关键实验室自动化的收购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.