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

Georges Belfort等rensselaer理工学院“NIRT：作为生物技术纳米开关的肠蛋白：将分子建模与生物物理和遗传方法联系起来”这是一个为期四年的跨学科、多研究者、多机构的提案，重点研究一种称为肠蛋白的自催化自我加工蛋白质，该蛋白质将被改编为纳米开关。本研究项目的目标是确定在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分子纳米开关，该开关显示出用于功能基因组学和蛋白质组学的理想特性。这项工作最终将使用纳米开关在流体芯片平台上进行蛋白质分离。研究方法将包括结合经典分子动力学和量子从头算与生物物理和遗传方法。经典计算和量子计算将共同提供分子水平的反应机制见解，以帮助确定对裂解反应至关重要的氨基酸，并指导位点定向诱变朝着更小、更快的裂解和特异性可控突变体的发展。将这一知识体系与生物物理测量和通过诱变获得的性能改进相结合，将获得具有纳米生物技术应用所需特性的分子纳米开关。具体目标是：1。利用经典分子动力学方法阐明在pH、温度等条件下剪接接点处关键氨基酸、盐分子和封闭水分子在剪接断裂过程中的作用、三维空间位置和运动，然后利用这些结果进行量子从头算计算，确定可能的键断裂位点作为pH和温度漂移的函数。利用中子交换和时间分辨电喷雾电离质谱（ESI-MS）、荧光共振能量转移（FRET）测量裂解反应过程中的内部构象动力学，并利用圆二色（CD）和傅里叶变换红外（FTIR）光谱测量裂解过程中的二级结构变化。3. 利用上面1和2的结果，以及内部蛋白的x射线晶体结构，指导内部蛋白的特定诱变，以获得具有减小尺寸，增加切割速率和替代触发机制的切割突变体和切割肽。此外，将随机诱变与遗传选择方案和噬菌体展示相结合，选择具有理想特性的其他衍生物。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。更广泛的影响：提出的方法是新颖的，并利用了RPI和沃兹沃思中心教师的人才，以一种新的协同方式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（物理学、化学工程和遗传学/生物化学），并建立在以前的跨机构成功开发具有生物技术有用特征的内部衍生物的基础上。通过与工业界和其他学术实验室的合作，可控纳米开关的成功开发将对蛋白质组学（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学科的培训，包括分子建模、生物物理表征、生化工程和分子遗传学。新的学生课程和基于网络的儿童视觉学习已经开始，并将在这个项目中得到强调。我们还参与了一个名为“新视野”（new Visions）的新项目，该项目旨在吸引来自社会经济弱势群体的高中生。综上所述，该研究将对推进蛋白质加工的发现和理解产生广泛的影响，同时促进纳米尺度科学与工程的教学、培训和学习。该项目由CTS/ENG，生物学和INT NSF组织支持。

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.