Experimental and Theoretical Studies of Charge-Charge Interactions in Proteins

本项目的目的是进一步了解电荷-电荷相互作用在决定蛋白质和酶的热力学和动力学稳定性中的作用。 实验工作将使用几种模型蛋白质和酶进行，每种模型蛋白质和酶都独特地适合于要解决的问题。 将比较冷休克蛋白（CspB-Bs和CspB-TB）、泛素（UBQ）、人羧肽酶原A2的活化结构域（ADA 2 h）、人腱生蛋白的纤连蛋白III型结构域（Tnf III）、人酰基磷酸酶（ACPh）和人U1 A蛋白的N-末端RNA结合结构域（U1 A）的合理稳定变体的折叠动力学与相应的野生型蛋白。 要解决的主要问题是，是否有一个主要的动力学机制的稳定蛋白质与优化的表面电荷-电荷相互作用。 还将研究单个结构域的稳定性对模型两结构域蛋白质的功能的作用，酵母3-磷酸甘油酸激酶（PGK），其在催化过程中经历大的构象变化。 将解决的主要问题是，是否合理优化的表面电荷-电荷相互作用的个别领域可以做不影响PGK的整体活动。 最后，蛋白质的稳定性在支持酶的活性在低温下的作用将进行研究，使用的模型酶，S-腺苷甲硫氨酸脱羧酶（METDC）从嗜冷，嗜温和嗜热生物的比较研究。 这项研究工作将使用合理的蛋白质工程和设计的方法来调节嗜冷，嗜温和嗜热的METDC蛋白的稳定性。 这种多学科的方法将能够深入了解与蛋白质稳定性相关的适应机制。 为此，结合各种方法，如比较序列分析，计算建模，蛋白质设计，一系列生物物理方法，（差示扫描量热法、等温滴定量热法、圆二色性光谱法、荧光光谱法、NMR光谱法、分析超离心法、动态光散射法、停止流动）和生物化学方法来表征导致模型蛋白质和酶的稳定性调节的生物物理机制和结构决定因素。 该项目将通过独特地结合各种生物化学，生物物理和计算工具，对蛋白质稳定化的机制进行系统研究。 因此，预计从这些实验中积累的知识将为未来研究这些根本性的重要问题奠定基础，这些问题对许多不同领域具有广泛的影响，包括开发下一代生物传感器，环境友好型催化剂和稳健的生物材料。 PI积极参与研究生和本科生教育和课程开发。 此外，各级学生（高中，本科和研究生）将直接参与PI实验室所有研究工作的计算和实验方面。

The objective of this project is to further understand the role of charge-charge interactions in determining the thermodynamic and kinetic stability of proteins and enzymes. The experimental work will be performed with several model proteins and enzymes, each uniquely suited for the questions to be addressed. The folding kinetics of rationally stabilized variants of cold shock proteins (CspB-Bs and CspB-TB), ubiquitin (UBQ), the activation domain of human procarboxypeptidase A2 (ADA2h), the fibronectin type III domain of human tenascin (TnfIII), human acylphosphatase (ACPh), and the N-terminal RNA-binding domain of human U1A protein (U1A) will be compared with the corresponding wild type proteins. The major question to be addressed is whether there is a predominant kinetic mechanism of stabilization for proteins with optimized surface charge-charge interactions. The role of the stability of individual domains for the function of a model two-domain protein, the yeast 3-phosphoglycerate kinase (PGK), which undergoes large conformational changes during the catalysis, will also be studied. The major question that will be addressed is whether the rational optimization of surface charge-charge interactions of individual domains can be done without affecting the overall activity of PGK. Finally, the role of protein stability in supporting the activity of enzymes at low temperature will be investigated using comparative studies of a model enzyme, S-adenosylmethionine decarboxylase (AdoMetDC) from psychrophilic, mesophilic and thermophilic organisms. This research effort will use methods of rational protein engineering and design to modulate stabilities of the psychrophilic, mesophilic and thermophilic AdoMetDC proteins. Such multidisciplinary approaches will enable to gain insights into the adaptation mechanisms that are related to protein stability. To this end, a combination of various methods such as comparative sequence analysis, computational modeling, protein design, a battery of biophysical methods (differential scanning calorimetry, isothermal titration calorimetry, circular dichroism spectroscopy, fluorescence spectroscopy, NMR spectroscopy, analytical ultracentrifugation, dynamic light scattering, stopped-flow) and biochemical methods to characterize the biophysical mechanisms and structural determinants that lead to modulation of the stability of model proteins and enzymes will be used. This project will provide a systematic study of the mechanism of protein stabilization by uniquely combining a variety of biochemical, biophysical and computational tools. As such, it is expected that the knowledge accumulated from these experiments will lay the foundation for future studies of these fundamentally important issues with broad implications for many different areas including the development of the next generation of biosensors, environmentally friendly catalysts and robust biomaterials. The PI is actively involved in graduate and undergraduate education and curriculum development. In addition, students at all levels (high-school, undergraduate and graduate) will directly participate in both the computational and experimental aspects of all research efforts in the PIs laboratory.