The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. The most successful approaches to date are based on modulating gene production via an inducible coupling to known transcriptional effectors. However, the current set of useful regulatory elements lacks sufficient diversity to advance the abovementioned biotechnologies. Accordingly, the invention of novel higher-order biosensing proteins that modulate gene expression will catalyze a revolution in how we conduct basic and applied research over the next decade. The challenge now is how to translate knowledge regarding allosteric communication?a hallmark of biosensing?into a rational design protocol for cooperative function. Accordingly, the objective of this study is to establish an innovative rational design algorithm for creating higher-order biosensing elements that are highly sensitive and orthogonal to native functions. The novel molecular switches generated in the study will become the foundation of exciting technologies in natural and non-natural systems alike. To accomplish this objective, bio-inspired engineering principles that involve both experimental and computational strategies with feedback between the two will be employed. The technology developed in this proposal will exist at the intersection of engineering, life science and information technology. Thus, this research will advance both engineering and life sciences via the creation of not only novel but useful biosensing elements in addition to addressing complementary scientific questions.
Broader Impacts: Establishing an integrated multidisciplinary program that is complementary to the aforesaid research initiative, while promoting diversity and increasing the involvement of underrepresented groups in biological engineering and science, will require the development of three underpinning and reciprocal criteria, namely: i) articulating the overarching goals of this research program and expressed interest in including underrepresented groups in these efforts to the broader community; ii) establishing a rigorous training initiative at both the undergraduate and graduate level; and iii) developing the necessary infrastructure to support these efforts. Likewise, this program will exist within the context of the mission goals and resources of the University. In turn, this effort will pave the way toward catalyzing a cultural change in undergraduate and graduate education, for students, faculty, and the University, by establishing an innovative model for promoting diversity and training in a fertile environment in the context of research that transcends traditional disciplinary boundaries. This initiative will contribute to the development of a diverse engaged science and engineering workforce, building a firm foundation for a lifetime of contributions to research, education and their integration.
The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. The most successful approaches to date are based on modulating mRNA synthesis via an inducible coupling to transcriptional effectors. However, the current set of useful regulatory elements lacks sufficient diversity to advance the abovementioned biotechnologies. Accordingly, the invention of novel higher-order biosensing proteins that modulate gene expression will catalyze a revolution in how we conduct basic and applied research over the next decade. The challenge now is how to translate our understanding of allosteric communication—a hallmark of biosensing—into a rational design protocol for cooperative function. Here we developed a protein design strategy for the creation of non-natural transcriptional regulatory proteins. To facilitate the creation of novel molecular switches we leveraged: i) our understanding of the structure and function of a prototypic regulatory element—i.e., lac repressor (LacI); ii) our ability to engineer allosteric routes; and iii) our ability to create specific and well ordered protein-protein interactions that are complementary to protein sensing and response functions. Significance and impact on the state-of-the-art: Here we established a rational design strategy to engineer novel protein-based regulatory elements that will become the foundation of exciting technologies in natural and non-natural systems alike. In the decade following this study our design algorithm will be used to create higher-order devices produced for use in high-throughput biosensing applications and monitoring biological structure interaction (e.g., protein-protein, protein-DNA and protein-ligand interactions). In addition, the technology developed here can be used to systematically identify and resolve critical gaps in our understanding of cooperative function. Looking forward, this advance will lead to future projects in which we will use our computational-experimental algorithm to create complex interfaces for novel molecular sensing devices—e.g., light-inducible or pH responsive molecular switches. Research Publications Understanding thermal adaptation of enzymes through the multistate rational design and stability prediction of 100 adenylate kinases. Howell, S.C., Inampudiand, K.K., Bean, D.P. and Wilson, C.J. (2014) Structure, PMID: 24361272 Rational Protein Design: Developing Next Generation Biological Therapeutics and Nanobiotechnological Tools. Wilson, C.J. (2014) WIREs Nanomedicine & Nanobiotechnology PMID: 25348497 Engineering alternate cooperative-communications in the lactose repressor protein scaffold, Protein engineering, design & selection. Meyer, S., Ramot, R., Kishore Inampudi, K., Luo, B., Lin, C., Amere, S., and Wilson, C. J. (2013) Protein Engineering Design & Selection 26, 433-443. PMID: 23587523 Lactose repressor experimental folding landscape: fundamental functional unit and tetramer folding mechanisms. Ramot, R., Kishore Inampudi, K., and Wilson, C. J. (2012) Biochemistry 51, 7569-7579.
