Insufficient stability of proteins is a fundamental problem that limits their applications. Several strategies have been reported to improve protein stability, but these approaches are specific to certain proteins or may compromise a protein's intrinsic properties. The previous studies from the PI's lab demonstrated that insertion into a thermophilic chaperoning protein can improve the thermal stability of various enzymes. Importantly, stabilization of enzymes in this manner caused no compromise in enzymatic activities and expression levels. The objectives of this project are 1) to reveal design principles for protein stabilization by insertion into a thermophilic chaperoning protein, 2) to determine the optimal insertion sites for effective protein stabilization, and 3) to further demonstrate the generality of this approach. To achieve these objectives, the PI and his group will apply their stabilization method to an enzyme, industrially important and highly suitable for the mechanistic study on stabilization. Rational and combinatorial protein engineering tools as well as experimental, theoretical and computational characterization methods will be employed. Quantitative and comprehensive evaluation of thermodynamic and kinetic stability will also be carried out.
BROADER IMPACT When successfully completed, outcomes from the project will offer a foundation to establish a simple, highly effective and potentially general "plug and play" molecular platform for stabilizing various proteins while maintaining their intrinsic properties. Such a stabilization method would greatly benefit many areas where insufficient stability of proteins limits their applications. The integration of research, education and outreach will result in the advancement of the educational and professional development of K-12, undergraduate and graduate students as well as teachers in a multidisciplinary area that overlaps biochemistry, biophysics, protein engineering and computational modeling. The proposed education and outreach plans, which are tightly integrated with the research plan, will focus on expanding the participation of underrepresented minority students in the study of science and engineering, and foster students' creativity and problem solving ability.