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

抗体代表着治疗人类疾病的一类日益重要的分子。人们对控制这些大分子的相行为非常感兴趣，从防止它们的缩合(在高浓度治疗配方中)到促进它(用于蛋白质结晶)。这个项目的目的是阐明抗体的自结合和相行为如何通过改变抗体表面暴露于溶剂的环来以系统的方式进行调节。重要的是，人们对溶剂暴露残基如何影响蛋白质的自结合和相行为知之甚少，我们认为这是由于：i)缺乏仅在其表面具有序列变化的相关同源蛋白质文库；ii)不能在不破坏其折叠结构的情况下对蛋白质表面进行显著的、系统的改变；智能优点：在这个项目中，假设小抗体(12kD)表面的溶剂暴露的多肽环(5-20个残基)可以被设计成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无关蛋白质的相应溶液行为。此外，据推测，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巨大变化具有高度的耐受性，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、胶体和界面科学以及分子生物学和生物化学的生物物理分析方面的独特优势的基础上。在具体目标1中，计划研究单一溶剂暴露抗体环中的序列变化(疏水性、电荷、长度和灵活性)对天然抗体自结合(根据渗透第二维里系数)和相行为的影响。接下来，在特定的目标2中，他们建议阐明在目标1中研究的抗体环影响天然抗体自身相互作用的机制，并确定这些环序列的子集是否能够通过环嫁接来调节无关蛋白质的相应热力学行为。然后，在具体的目标3中，他们建议确定在目标1和目标2中研究的抗体变体的独特的非天然溶解度(在瞬时热处理后)是否可以与其天然蛋白质自身相互作用的测量相关联，类似于将吸引人的蛋白质自身相互作用与天然折叠蛋白质的低溶解度(和更高的蛋白质结晶可能性)联系起来的“结晶槽”概念。最后，在特定的目标4中，他们建议阐明如何通过对每个环残基对不希望的抗体自结合行为的贡献进行全面的突变和自我相互作用分析，来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的全面的突变和自我相互作用分析，将与AIMS 1-3中研究的抗体密切相关的抗体(在其天然和非天然状态下)设计成抗聚集的抗体。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，对于防止疾病相关蛋白聚集、制造潜在更稳定的治疗性蛋白以及操纵蛋白质晶体的组装具有广泛的意义。在教育方面，PIS致力于通过开设新课程(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，从而使伦斯勒的课程现代化。他们还致力于通过两项努力向未被充分代表的少数族裔和其他弱势群体大力宣传：i)在一所小学的四年级科学推广计划，午餐减少(约80%)，非裔美国人(约40%)的学生专注于分子，使用动画片和动手活动，以使这些学生在早期对科学感兴趣；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年级推广计划，让这些学生参与发现和开发治疗性抗体的过程，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.