SGER:Developing New Methods Based on Folding and Unfolding Pathways

提案题目：SGER开发基于折叠和展开路径的新方法。项目编号：ctis -0621216项目负责人：Anubhav Tripathi，机构：Brown University智力优势：高分辨率分离方法旨在将复杂的大分子混合物分解为单个组分，以便随后识别大分子表达模式，并在更广泛的范围内获得对生物系统调控的基本理解。常见的分离策略有：(1)电泳，根据电泳迁移率和等电点分离大分子；(2)色谱法，根据迁移率、疏水性和/或亲和性分离大分子。由于这些策略要么使用完全折叠的大分子，要么使用完全变性的大分子，因此很难分辨具有相似迁移率、等电点和/或疏水性的大分子。蛋白质和rna是大分子的两个例子，它们根据水环境的质量采取不同的折叠-展开结构，从而破坏疏水相互作用或改变分子上的净电荷，引起静电排斥或破坏一些氢键。在分离与纯化科学中，需要一个新的研究方向，使蛋白质和rna等大分子在特定的缓冲条件下采用不同的折叠-展开途径。在这些条件下，两个分子可以根据其迁移率或吸附/解吸特性的“增强”差异进行分离。研究者最近证明了乳球蛋白的两种异构体在一定的缓冲条件下具有不同的构象结构。在本次SGER提案中，PI已申请资金进行初步实验，以证明在特定的缓冲条件下，由于构象的差异，两种同工异构体具有不同的迁移率和不同的吸附等温线。此外，迁移率和吸附行为的差异可以用来分离两种异构体，使用(a)电泳和(b)吸附在微芯片几何形状的球体填充床上。SGER的结果将验证伴随部分展开的蛋白质特性变化可能用于分离的说法。如果成功，PI预计SGER提案将导致更详细的CAREER提案，其中蛋白质折叠和展开途径的亲密细节将在精心制作的实验和教学计划中进行建模和表征。研究的更广泛影响是具有类似流动性的大分子的量化，鉴于它们在突变、不适当表达或功能的病理状态（例如朊病毒和各种癌症）中的核心作用，这一点变得非常重要。在未来，这一领域的研究项目可能会提供一种质量控制过程来测试重组人蛋白的纯度和稳定性。该程序还可能为制药行业提供一种筛选方法，可以测试数千种配体分子的结合能力，以抑制相关靶酶或蛋白质的活性。在这个SGER中进行的基础实验将探索这些问题，并将为跨学科研究提供一些新的主题。该课程将热力学、动力学和输运现象的核心技能与分离和净化科学相结合。

Abstract Proposal Title: SGER Developing New Methods Based on Folding and Unfolding Pathways. Proposal Number: CTS-0621216 Principal Investigator: Anubhav Tripathi, Institution: Brown University Intellectual Merit: High resolution separation methodologies aim to resolve complex mixtures of macromolecules into individual components for subsequent identification of macromolecular expression patterns and, on a broader scale, gain a fundamental understanding of biological system regulation. Common separation strategies are: (1) electrophoresis in which macromolecules are separated based on electrophoretic mobility and isoelectric point, and (2) chromatography in which the macromolecules are separated based on mobility, hydrophobicity and/or affinity. Since these strategies use either fully folded or fully denatured macromolecules, it is extremely difficult to resolve macromolecules with similar mobilities, isoelectric points and/or hydrophobicities. Proteins and RNAs are two examples of macromolecules which adopt different folded-unfolded structures based on the quality of aqueous environment which can disrupt the hydrophobic interactions or alter the net charge on the molecule causing electrostatic repulsion or disruption of some hydrogen bonds. A new research direction in separation and purification science, in which macromolecules such as proteins and RNAs can be targeted to adopt different folding-unfolding pathways under specific buffer conditions, is needed. Under these conditions, two molecules can be separated based on the "enhanced" difference in their mobility or adsorption/desorption characteristics. The investigator has recently demonstrated that two isoforms of lactoglobulin possess conformationally different structures under certain buffer conditions. In this SGER proposal, the PI has requested funding to perform preliminary experiments to demonstrate that under specific buffer conditions, due to differences in conformations, the two isoforms have different mobilities and different adsorption isotherms. Furthermore, differences in mobilities and adsorption behavior can be used to separate the two isoforms using (a) electrophoresis and (b) adsorption on a packed bed of spheres in microchip geometry. The results from this SGER will validate the claim that the changes in protein properties that accompany partial unfolding might be utilized for separation. If successful, the PI anticipates that this SGER proposal will lead to a more detailed CAREER proposal where the intimate details of protein folding and unfolding pathways will be modeled and characterized within a carefully crafted experimental and teaching plan. The broader impact of research is the quantification of macromolecules with similar mobilities that has become important in light of their central role in pathological states of mutations, improper expression or function (e.g. prion and various cancers). In the future, a research program in this area might offer a quality control process to test purity and stability of recombinant human proteins. The program also might offer a screening method for the pharmaceutical industry where binding ability of thousands of ligand molecules can be tested to inhibit the activity of a relevant target enzyme or protein. The fundamental experiments to be performed in this SGER will explore these issues and will provide several host new topics for interdisciplinary research. The educational program couples core skills of thermodynamics, kinetics and transport phenomenon to separation and purification science.