NIRT:Intein Proteins as Nanoswitches for Biotechnology:Linking Molecular Modeling with Biophysical and Genetic Methods

NIRT：内含肽作为生物技术的纳米开关：将分子建模与生物物理和遗传学方法联系起来

• 批准号: 0304055
• 负责人: Georges Belfort
• 金额: $ 121.52万
• 依托单位: Rensselaer Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0304055&HistoricalAwards=false
• 关键词: NIRT; Intein; Proteins; Nanoswitches; Biotechnology

Georges Belfort et al.Rensselaer Polytechnic Institute"NIRT: Intein Proteins as Nanoswitches for Biotechnology: Linking Molecular Modeling with Biophysical and Genetic Methods"This is a four-year cross-disciplinary, multi-investigator, multi-institutional proposal focused on an autocatalytic self-processing protein called an intein, which will be adapted as a nanoswitch. The goals of this research project are to determine the underlying principles of the splicing and cleavage reactions that occur during protein processing and to use this understanding to design a molecular nanoswitch that exhibits desirable properties for use in functional genomics and proteomics. This work will culminate in the use of the nanoswitch to perform protein separation on a fluidics chip platform. The research approach will involve combining classical molecular dynamics and quantum ab initio calculations with biophysical and genetic methods. Together classical and quantum calculations will provide molecular-level insights into the reaction mechanism to help identify amino acids critical to the cleavage reaction and guide the site-directed mutagenesis towards development of smaller, faster cleaving and specifically controllable mutants. Combining this body of knowledge with that from biophysical measurements and performance improvements obtained through mutagenesis, molecular nanoswitches with desirable characteristics for applications in nano-biotechnology will be obtained. The specific aims are to: 1. Use classical molecular dynamics to elucidate the role, the three-dimensional spatial location and the movement of critical amino acids, salt molecules and occluded water molecules in the intein cleavage process during excursions in pH, temperature and other conditions at the splice junctions, and then use these results for quantum ab initio calculations to determine the likely bond cleavage sites as a function of excursions in pH and temperature.2. Measure intein conformational dynamics during the cleavage reaction using euterium-exchange and time-resolved electrospray ionization-mass spectrometry (ESI-MS), Fluorescence Resonance Energy Transfer (FRET), and measure the secondary structural changes during cleavage using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. 3. Use the results from 1 and 2 above, and X-ray crystallographic structures of inteins, to guide specific mutagenesis of inteins to obtain cleavage mutants and cleavage peptides with reduced size, increased cleavage rates, and alternative trigger mechanisms. In addition, combine random mutagenesis with genetic selection schemes and phage display to select for additional derivatives with desirable characteristics. 4. Test newly developed molecular nanoswitches on a fluidics-chip platform for one-step protein recovery for a proteomics application.Broader Impacts:The proposed approach is novel and uses talents of RPI and Wadsworth Center faculty in a new and synergistic way to address a significant bio- and nano-technology problem. The proposed work is cross-disciplinary (physics, chemical engineering, and genetics/biochemistry) and builds upon previous cross-institutional success developing intein derivatives with useful characteristics for biotechnology. Successful development of controllable nanoswitches will have impact on proteomics (bioseparations on fluidics chip platform) and medicine (biosensors and drug delivery) through collaboration with industry and with other academic labs.Graduate and undergraduate students will be exposed to interdisciplinary training that spans molecular modeling, biophysical characterization, biochemical engineering and molecular genetics. New curricula for students and web-based visual learning for children has been initiated and will be emphasized in this project. We are also involved in assisting a new program, New Visions, which is interested in attracting high-school students from economically disadvantaged sections of society.In summary, the proposed research will have a broad impact on advancing discovery andunderstanding of protein processing while promoting teaching, training and learning in nanoscale science and engineering. The project is supported by CTS/ENG, Biology, and INT NSF organizations.

Rensselaer理工学院“NIRT：作为生物技术的纳米开关的内含子蛋白质：将分子建模与生物物理和遗传方法联系起来”这是一项为期四年的跨学科、多研究者、多机构的提案，重点是一种名为内含子的自动催化自我处理蛋白质，它将被改造为纳米开关。本研究项目的目标是确定蛋白质加工过程中发生的剪接和切割反应的基本原理，并利用这种理解来设计一种显示出在功能基因组学和蛋白质组学中使用的理想特性的分子纳米开关。这项工作将最终使用纳米开关在流体芯片平台上进行蛋白质分离。研究方法将包括将经典分子动力学和量子从头计算与生物物理和遗传学方法相结合。经典和量子计算将在分子水平上提供对反应机制的见解，以帮助识别对切割反应至关重要的氨基酸，并引导定点突变朝着开发更小、更快切割和特定可控突变体的方向发展。将这些知识与生物物理测量和通过诱变获得的性能改进相结合，将获得在纳米生物技术中应用的具有理想特性的分子纳米开关。其具体目的是：1.利用经典分子动力学来阐明在pH、温度等条件下，剪接连接处的内切过程中关键氨基酸、盐分子和封闭水分子在内切过程中的作用、三维空间位置和运动，然后利用这些结果进行量子从头计算，以确定可能的键裂解位置作为pH和温度偏移的函数。利用电子交换和时间分辨电喷雾电离-质谱仪(ESI-MS)、荧光共振能量转移(FRET)测量切割过程中内含子的构象动力学，利用圆二色谱(CD)和傅里叶变换红外光谱(FTIR)测量切割过程中二级结构的变化。3.利用上述1和2的结果以及内含子的X射线晶体结构，指导内含子的特异性诱变，以获得尺寸更小、切割速度更快、触发机制可选的切割突变体和切割多肽。此外，将随机诱变与遗传选择方案和噬菌体展示相结合，以选择具有所需特性的额外衍生物。4.在流体芯片平台上测试新开发的分子纳米开关，用于蛋白质组学应用的一步蛋白质回收。广泛影响：建议的方法是新颖的，以一种新的和协同的方式利用RPI和Wadsworth Center的人才来解决一个重要的生物和纳米技术问题。拟议的工作是跨学科的(物理、化学工程和遗传学/生物化学)，并建立在以前开发具有生物技术有用特征的INTIN衍生物的跨机构成功的基础上。可控纳米开关的成功开发将通过与工业界和其他学术实验室的合作，对蛋白质组学(流体芯片平台上的生物分离)和医学(生物传感器和药物输送)产生影响。研究生和本科生将接受跨越分子建模、生物物理表征、生物化学工程和分子遗传学的跨学科培训。针对学生的新课程和针对儿童的基于网络的视觉学习已经启动，并将在本项目中得到强调。我们还参与了一个名为新视野的新项目，该项目旨在吸引来自经济困难阶层的高中生。总而言之，拟议的研究将对促进蛋白质加工的发现和理解产生广泛影响，同时促进纳米科学和工程的教学、培训和学习。该项目得到了CTS/ENG、生物学和INT NSF组织的支持。