| The ability to engineer novel binding proteins is important in molecular biology, biotechnology and medicine. Proteins engineered to interact with other molecules with high affinity and specificity are used for a wide range of applications including the labeling and detection, purification, and functional perturbation of target molecules. Because of these and other important practical applications, there is a strong need to develop systems for engineering novel binding proteins quickly and effectively. To date, antibodies have been the most widely used class of binding proteins for most applications. However, non-antibody platforms for engineering novel binding proteins have emerged as attractive alternatives in the past 10-15 years. Our laboratory has pioneered the use of one such alternative scaffold, the fibronectin type III domain (FN3). We call the novel binding proteins produced based on this scaffold, "monobodies". Since the establishment of the monobody system 12 years ago, it has emerged as one of the most successful and widely used alternative scaffold systems. High-affinity monobodies have been successfully generated to a wide array of target molecules and have been used in a variety of practical applications. However, despite this success, no detailed structural or mechanistic information has been obtained for how monobodies achieve these activities. As a result, critical evaluation of current monobody engineering strategies and the development of new strategies has remained a difficult task and our understanding of molecular recognition in the monobody system has remained very limited. In this dissertation, I seek to gain detailed structural and mechanistic characterization of monobodies in order to improve our engineering abilities and advance our understanding of molecular recognition in this important system. I have used this approach to investigate the roles of amino acid diversity and conformational diversity in the construction monobody-target interactions. The findings here have led to improved strategies for engineering novel binding proteins using the monobody system and carry implications for engineering strategies in other systems as well. Additionally, these findings help to illuminate the roles that amino acid diversity and conformational diversity play in protein-protein interactions more generally. Overall, the work here demonstrates the power of detailed structural and mechanistic characterization of engineered binding proteins in advancing our ability to engineer novel binding proteins and furthering our understanding of the factors governing molecular recognition. |
