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。实验工作将用几种模型蛋白质和酶进行，每一种都独特地适合要解决的问题。将合理稳定的冷休克蛋白（CspB-Bs和CspB-TB）、泛素（UBQ）、人原羧肽酶A2 （ADA2h）激活域、人张力蛋白纤维连接蛋白III型域（TnfIII）、人酰基磷酸酶（ACPh）和人U1A蛋白n端rna结合域（U1A）的折叠动力学与相应的野生型蛋白进行比较。要解决的主要问题是，是否存在一种主要的动力学机制来稳定具有优化表面电荷-电荷相互作用的蛋白质。酵母3-磷酸甘油酸激酶（酵母3-磷酸甘油酸激酶，PGK）是一种典型的双结构域蛋白，在催化过程中会发生巨大的构象变化，而单个结构域的稳定性对其功能的作用也将被研究。要解决的主要问题是，是否可以在不影响PGK整体活性的情况下，合理优化单个结构域的表面电荷-电荷相互作用。最后，通过对一种模型酶——s -腺苷蛋氨酸脱羧酶（AdoMetDC）的比较研究，研究了蛋白质稳定性在低温下支持酶活性的作用。该模型酶来自嗜冷、中温和嗜热生物。本研究将采用合理的蛋白质工程和设计方法来调节嗜冷、中温和嗜热AdoMetDC蛋白的稳定性。这种多学科的方法将使我们能够深入了解与蛋白质稳定性相关的适应机制。为此，结合多种方法如比较序列分析、计算建模、蛋白质设计、电池生物物理方法(差示扫描量热法、等温滴定量热法、圆二色光谱、荧光光谱、核磁共振光谱、分析超离心、动态光散射、将使用停止流动和生化方法来表征导致模型蛋白质和酶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化，生物物理和计算工具，对蛋白质稳定机制进行系统研究。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重要问题奠定基础，并对许多不同领域产生广泛影响，包括下一代生物传感器的开发，环境友好型催化剂和强大的生物材料。PI积极参与研究生和本科教育以及课程开发。此外，各级学生（高中、本科和研究生）将直接参与pi实验室的所有研究工作的计算和实验方面。