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CAREER:Experimental Studies of Protein Thermodynamics Facilitated by NMR with Reverse Micelles

职业：反胶束核磁共振促进蛋白质热力学实验研究

基本信息

批准号：
1942957
负责人：
Nathaniel Nucci
金额：
$ 74.94万
依托单位：
Rowan University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1942957&HistoricalAwards=false
关键词：
CAREER Experimental Studies Protein Thermodynamics

项目摘要

Though all cells are composed primarily of water, the environment inside the cell is quite heterogeneous, containing nanoscale spaces created by membrane-bound compartments and by high concentrations of large molecules including nucleic acids (DNA/RNA) and proteins. Generally, studies of proteins have been performed in dilute solutions because the cellular environment is too complex for most experimental approaches, thus the effects of this environment on protein structure and function remains poorly understood. This project uses nanobubbles, called reverse micelles, to mimic the cellular environment so that the influence of spatial restriction and interfacial interactions on proteins can be experimentally dissected. These experimental measurements are being compared to computer simulations of proteins inside these nanobubbles to produce a unified view of environmental effects on proteins. This approach is expected to provide a foundation for developing improved predictive models for protein function inside cells, particularly when the function of the protein is related to its structural stability. These studies are integrated with experiential education and mentorship of undergraduate students in the laboratory and the classroom including the development of a new research-based laboratory for an upper-level biophysics course aimed at helping students learn science by doing science. The project is designed to offer research experience to these students in a fashion that scales the complexity of the experiments and modeling as the students advance through their undergraduate education.In recent years, the relevance of the complex intracellular milieu in modulating protein structural stability and function has become clear. As a result, the paucity of predictive models for these effects represents an important biophysical knowledge gap. This research program combines nuclear magnetic resonance (NMR) with reverse micelle (RM) encapsulation of proteins to measure previously challenging thermodynamic environmental influences on protein structure and function. RMs are surfactant-stabilized complexes that spontaneously organize to encapsulate a nanoscale bubble of water in a nonpolar solvent. RMs have been shown to facilitate study of proteins by NMR including the ability to directly modulate their structural stability by varying the size of the bubble or the nature of the water-surfactant interface. This program pursues three specific avenues of inquiry to provide novel insight on 1. Weak electrostatic and hydrophobic forces between proteins and interfaces, 2. The magnitude of the hydrophobic effect, and 3. The effect of excluded volume on protein dynamics and disorder. Using a suite of model proteins and the tumor suppressor protein p53, which contains both highly stable folded domains and intrinsically disordered regions, these studies harness the tunability of reverse micelle systems to dissect the environmental thermodynamic driving forces of protein structure and quantify their differential contributions in unprecedented detail. These studies are integrated with an upper-division molecular biophysics undergraduate course via a research-based learning experience in which students design, execute, and report a novel study on the impact of crowding molecules on protein structural stability. The practical experience gained is reinforced through a mentorship program that matches students from underrepresented groups with STEM professionals (mentors) in their field of interest, thereby aiming to increase the percentage of underrepresented STEM graduates that successfully pursue STEM careers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

虽然所有的细胞主要由水组成，但细胞内的环境是相当不均匀的，包含由膜结合区室和高浓度的大分子（包括核酸（DNA/RNA）和蛋白质）创造的纳米级空间。一般来说，蛋白质的研究是在稀溶液中进行的，因为细胞环境对于大多数实验方法来说太复杂了，因此这种环境对蛋白质结构和功能的影响仍然知之甚少。该项目使用称为反胶束的纳米气泡来模拟细胞环境，以便可以通过实验解剖空间限制和界面相互作用对蛋白质的影响。这些实验测量正在与这些纳米气泡内蛋白质的计算机模拟进行比较，以产生环境对蛋白质影响的统一观点。这种方法有望为开发细胞内蛋白质功能的改进预测模型提供基础，特别是当蛋白质的功能与其结构稳定性相关时。这些研究与实验室和课堂上的体验式教育和本科生导师相结合，包括为高级生物物理课程开发一个新的研究型实验室，旨在帮助学生通过做科学来学习科学。该项目旨在为这些学生提供研究经验，随着学生本科教育的发展，实验和建模的复杂性将逐步增加。近年来，复杂的细胞内环境在调节蛋白质结构稳定性和功能方面的相关性已经变得清晰。因此，缺乏这些影响的预测模型是一个重要的生物物理知识差距。该研究计划将核磁共振（NMR）与蛋白质的反胶束（RM）封装相结合，以测量先前具有挑战性的热力学环境对蛋白质结构和功能的影响。RM是表面稳定的复合物，其自发组织以在非极性溶剂中封装纳米级水气泡。RM已被证明有助于通过NMR研究蛋白质，包括通过改变气泡的大小或水-表面活性剂界面的性质直接调节其结构稳定性的能力。该计划追求三种具体的调查途径，以提供1.蛋白质和界面之间的弱静电力和疏水力，2。疏水效应的大小，和3。排除体积对蛋白质动力学和紊乱的影响。使用一套模型蛋白质和抑癌蛋白p53，其中包含高度稳定的折叠结构域和内在的无序区域，这些研究利用反胶束系统的可调性解剖蛋白质结构的环境热力学驱动力，并量化其差异贡献前所未有的细节。这些研究通过基于研究的学习体验与高年级分子生物物理本科课程相结合，学生设计，执行和报告拥挤分子对蛋白质结构稳定性影响的新研究。获得的实践经验通过导师计划得到加强，该计划将来自代表性不足的群体的学生与他们感兴趣的领域的STEM专业人员（导师）相匹配，该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查进行评估，被认为值得支持的搜索.