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

职业：蛋白质表面的循环工程，用于可调节的自缔合和相行为

• 批准号: 0954450
• 负责人: Peter Tessier
• 金额: $ 41.19万
• 依托单位: Rensselaer Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0954450&HistoricalAwards=false
• 关键词: CAREER; Loop; engineering; protein; surfaces

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.

0954450 Tessier抗体代表了一类日益重要的治疗人类疾病的分子。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）。这个项目的目的是阐明如何抗体的自缔合和相行为可以通过改变抗体表面上的溶剂暴露环系统地调制。重要的是，关于溶剂暴露的残基如何影响蛋白质自缔合和相行为知之甚少，我们认为这是由于：i）缺乏仅在其表面上具有序列变化的相关同源蛋白质文库; ii）无法在不破坏其折叠结构的情况下对蛋白质表面引入显著的系统性改变;和iii）以可靠和快速的方式测量蛋白质自身相互作用的困难。智力优点：在这个项目中，它是假设，溶剂暴露的肽环，小抗体（12 kD）表面上的多个（5-20个残基）可以被工程化以调节天然和非天然折叠抗体的自缔合和相行为，并且这些环可用于通过环移植来控制不相关蛋白质的相应溶液行为。此外，据推测，具有明确定义的表面环的小抗体是用于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，而不改变它们的折叠稳定性。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之上。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异（疏水性、电荷、长度和柔性）对天然抗体自缔合（根据渗透第二维里系数）和相行为的影响。接下来，在具体目标2中，他们提出阐明目标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移植调节无关蛋白质的相应热力学行为。然后，在具体目标3中，他们建议确定独特的非天然溶解度是否目的1和2中研究的抗体变体（瞬时热处理后）（其不同之处仅在于它们的环序列）可以与它们的天然蛋白质自身相互作用的测量相关联，类似于将有吸引力的蛋白质自身相互作用与低溶解度联系起来的“结晶槽”概念（和增加的蛋白质结晶的可能性）。最后，在具体目标4中，他们提出阐明与目标1-3中研究的抗体密切相关的抗体如何易于聚集通过对每个环残基对不期望的抗体自缔合行为的贡献进行全面的突变和自相互作用分析，可以将环残基（在其天然和非天然状态下）改造为具有最小序列改变的抗聚集性。该项目深深植根于分子热力学和界面工程科学，对预防疾病相关蛋白质聚集，制造潜在的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意义。在教育方面，PI致力于使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，向代表性不足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，减少午餐（约80%）和非洲裔美国人（约40%）的学生，重点是使用动画片和动手活动来关注分子，以使这些学生从小就对科学感兴趣;以及ii）针对来自纽约首都区的农村城镇的缺乏高级科学课程的不同学生的12年级外展计划，该计划使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。