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

Rensselaer Polytechnic Institute, Troy NY

Investigators

Abstract

0954450 Tessier Antibodies 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.