Experimental and Theoretical Studies of Charge-Charge Interactions in Proteins

蛋白质中电荷相互作用的实验和理论研究

• 批准号: 0818419
• 负责人: George Makhatadze
• 金额: $ 103.37万
• 依托单位: Rensselaer Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0818419&HistoricalAwards=false
• 关键词: Experimental; Theoretical; Studies; Charge; Interactions

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.

该项目的目的是进一步了解电荷 - 电荷相互作用在确定蛋白质和酶的热力学和动力学稳定性中的作用。 实验性工作将使用几种模型蛋白和酶进行，每个蛋白质和酶都非常适合要解决的问题。 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人U1a蛋白（U1A）的RNA结合结构域将与相应的野生型蛋白进行比较。 要解决的主要问题是，是否存在具有优化的表面电荷相互作用的蛋白质稳定的主要动力学机制。 单个结构域在催化过程中经历大构象变化的酵母3-磷酸甘油酸激酶（PGK）的稳定性在催化过程中经历了较大构象变化的作用。 将要解决的主要问题是，是否可以在不影响PGK的整体活动的情况下完成表面充电 - 电荷相互作用的合理优化。 最后，将研究蛋白质稳定性在低温下支持酶活性中的作用，将使用基督，嗜嗜嗜性剂，嗜嗜嗜性和热嗜热生物的模型酶，S-腺苷甲硫代氨基氨酸脱羧酶（ADOMETDC）的比较研究进行研究。 这项研究工作将使用理性蛋白质工程和设计的方法来调节精神嗜，嗜嗜和嗜热的ADOMOMIC ADOMOMTDC蛋白的稳定性。 这种多学科方法将使能够深入了解与蛋白质稳定性相关的适应机制。 为此，各种方法的结合，例如比较序列分析，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，圆形二色镜检查，循环二色镜检查，浮动光谱，NMR光谱，NMR光谱范围，定制超出范围散射，动态散射，动态散射，动态散射，动态散射，动态化，动态散射，动态散射，动态散射，动态散射，将使用表征导致模型蛋白和酶稳定性调节的生物物理机制和结构决定因素的方法。 该项目将通过唯一结合多种生化，生物物理和计算工具来提供对蛋白质稳定机制的系统研究。 因此，预计从这些实验中积累的知识将为未来对这些根本重要问题的研究奠定基础，对许多不同领域的广泛影响，包括下一代生物传感器，环保催化剂和强大的生物材料的发展。 PI积极参与研究生和本科教育和课程发展。 此外，各个级别的学生（高中，本科和研究生）将直接参与PIS实验室所有研究工作的计算和实验方面。