CAREER: Loop engineering of protein surfaces for tunable self-association and phase behavior

抗体是治疗人类疾病的一类越来越重要的分子。人们对控制这些大分子的相行为非常感兴趣，从防止它们的冷凝（在高浓度治疗制剂中）到促进它（用于蛋白质结晶）。该项目的目的是阐明如何通过改变抗体表面的溶剂暴露环来系统地调节抗体的自结合和相行为。重要的是，我们对溶剂暴露残留物如何影响蛋白质自结合和相行为知之甚少，我们认为这是由于：i)缺乏相关的同源蛋白文库，仅在其表面上有序列变化；Ii)无法在不破坏蛋白质折叠结构的情况下对蛋白质表面进行重大、系统的改变；iii)难以以可靠和快速的方式测量蛋白质自身相互作用。智力优势：在这个项目中，假设在小抗体（12 kD）表面的溶剂暴露肽环（5-20个残基）可以被设计来调节天然和非天然折叠抗体的自结合和相行为，并且这些环可以通过环嫁接来控制不相关蛋白质的相应溶液行为。此外，假设具有明确表面环的小抗体是此类研究的有吸引力的模型蛋白，因为它们对环大小和组成的大变化具有高度耐受性，而不会改变其折叠稳定性。因此，为了验证这些假设，研究人员提出了四个具体目标，这些目标建立在我们在蛋白质溶液热力学、胶体和界面科学、分子生物学和生物化学的生物物理分析方面的独特优势之上。在Specific Aim 1中，计划研究单个溶剂暴露抗体环中序列变化（疏水性、电荷、长度和柔韧性）对天然抗体自结合（根据渗透第二病毒系数）和相行为的影响。接下来，在特异性目标2中，他们提出阐明在目标1中研究的抗体环影响天然抗体自相互作用的机制，并确定这些环序列的一个子集是否能够通过环嫁接来调节不相关蛋白质的相应热力学行为。然后，在特定目标3中，他们提议确定目标1和目标2中研究的抗体变体（仅在其环序列不同）的独特非天然溶解度（在瞬态热处理后）是否可以与其天然蛋白质自相互作用的测量相关联，类似于“结晶槽”概念，将有吸引力的蛋白质自相互作用与天然折叠蛋白质的低溶解度（和增加的蛋白质结晶可能性）联系起来。最后，在Specific Aim 4中，他们提出阐明如何通过对每个环残基对不良抗体自结合行为的贡献进行全面的突变和自相互作用分析，来阐明与Aims 1-3中研究的抗体密切相关的易于聚集的抗体（在其原生和非原生状态下）可以被设计成具有最小序列改变的聚集抗性。更广泛的影响：该项目深深植根于分子热力学和界面工程科学，对预防疾病相关的蛋白质聚集，制造潜在更稳定的治疗蛋白质以及操纵蛋白质晶体的组装具有广泛的影响。在教育方面，pi致力于伦斯勒的课程现代化，通过新课程（生物分子工程）和实验室实验（结晶）向本科生和研究生介绍分子水平的概念。他们还致力于通过两项努力向未被充分代表的少数民族和其他弱势群体提供强有力的外展服务：i)在一所小学开展四年级科学外展计划，其中很大一部分减少了午餐（~80%）和非洲裔美国人（~40%）的学生专注于分子，使用动画漫画和动手活动，让这些学生从小就对科学产生兴趣；ii)针对纽约首都地区农村城镇缺乏高级科学课程的不同学生的12年级拓展计划，让这些学生参与治疗性抗体的发现和开发过程，以鼓励他们在本科和研究生教育期间追求科学和工程的生物分子方面。

0954450TessierAntibodies represent an increasingly important class of molecules to treat human disease. There is significant interest in controlling the phase behavior of these macromolecules, ranging from preventing their condensation (in high concentration therapeutic formulations) to promoting it (for protein crystallization). The objective of this project is to elucidate how antibody self-association and phase behavior can be modulated in a systematic manner through alteration of solvent exposed loops on antibody surfaces. Importantly, little is known about how solvent exposed residues impact protein selfassociation and phase behavior, which we argue is due to: i) the lack of relevant homologous protein libraries with sequence variations only on their surface; ii) the inability to introduce significant, systematic alterations to protein surfaces without disrupting their folded structure; and iii) the difficulty in measuring protein self interactions in a reliable and rapid manner.Intellectual Merit: In this project it is postulated that solvent-exposed peptide loops (5-20 residues) on the surface of small antibodies (12 kD) can be engineered to regulate the self-association and phase behavior of both natively and non-natively folded antibodies, and that these loops can be used to control the corresponding solution behavior of unrelated proteins via loop grafting. Moreover, it is postulated that small antibodies with well-defined surface loops are attractive model proteins for such studies since they are highly tolerant to large changes in loop size and composition without altering their folding stability. Therefore, to test these hypotheses, the investigators propose four Specific Aims that build on our unique strengths in biophysical analysis of protein solution thermodynamics, colloidal and interface science, and molecular biology and biochemistry. In Specific Aim 1, it is planned to investigate the impact of sequence variations (hydrophobicity, charge, length and flexibility) in a single solvent exposed antibody loop on native antibody self association (in terms of the osmotic second virial coefficient) and phase behavior. Next, in Specific Aim 2, they propose to elucidate the mechanisms by which antibody loops studied in Aim 1 influence native antibody self-interactions, and to determine if a subset of these loop sequences are capable of regulating the corresponding thermodynamic behavior of unrelated proteins via loop grafting. Then, in Specific Aim 3, they propose to ascertain if the unique non-native solubilities (after transient heat treatment) of antibody variants studied in Aims 1 and 2 (which differ only in their loop sequences) can be linked to measurements of their native protein self interactions, akin to the "crystallization slot" concept that links attractive protein self interactions to low solubility (and increased likelihood of protein crystallization) of natively folded proteins. Finally, in Specific Aim 4, they propose to elucidate how an antibody closely related to those studied in Aims 1-3 that is aggregation prone (in both its native and nonnative states) can be engineered to be aggregation resistant with minimal sequence alteration through comprehensive mutational and self-interaction analysis of the contribution of each loop residue to the undesirable antibody self-association behavior.Broader Impacts: This project, deeply rooted in molecular thermodynamics and interfacial engineering science, has broad implications for preventing disease-associated protein aggregation, making potentially more stable therapeutic proteins, and manipulating assembly of protein crystals. In terms of education, the PIs are committed to modernizing their curricula at Rensselaer by introducing undergraduate and graduate students to molecular-level concepts through a new course (Biomolecular Engineering) and laboratory experiment(crystallization). They are also committed to strong outreach to underrepresented minorities and other disadvantaged peoples through two efforts: i) a 4th grade science outreach program in an elementary school with a significant fraction of reduced lunch (~80%) and African American (~40%) students focused on molecules using animated cartoons and hands-on activities to interest these students in science at an early age; and ii) a 12th grade outreach program to diverse students from rural towns in New York's Capital District lacking advanced science courses that involves these students in the process of discovery and development of a therapeutic antibody to encourage them to pursue biomolecular aspects of science and engineering during their undergraduate and graduate education.