Development and Application of Method to Study Protein Kinetic Stability

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Like a child strapped to a high-chair, certain proteins are trapped in their native structure, unable to transiently sample partially and globally unfolded conformations. This appears to be a method used by "mother nature" to protect some proteins from degradation or misfolding, either one of which would compromise the function of the protein and the well-being of the organism. Proteins that possess this property are described as being kinetically stable because, unlike most proteins, their long "shelf-life" and stability towards degradation is kinetically controlled by a slow unfolding rate. These proteins are trapped by a high energy barrier towards unfolding. The structural basis and biological significance of this property remains poorly understood. The investigators recently demonstrated a correlation between kinetic stability and a protein's resistance to the detergent sodium dodecyl sulfate (SDS), and subsequently developed a simple diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) method to identify kinetically stable proteins in complex mixtures (cell lysates). The goal of this project is to apply the D2D SDS-PAGE method in combination with mass spectrometry and proteomics analysis to a diverse organisms to explore the biological significance and pervasiveness of kinetic stability, and to develop a new method to quantify kinetic stability. Results of this project should lead to a better understanding of the structural basis of kinetic stability and its roles in nature. The outcome of the project will provide simple methods to facilitate the study of protein kinetic stability in mainstream biology labs. Finally, since proteins are being increasingly used in a variety of applications, including pharmaceuticals, renewable energy, and agriculture, this project could lead to the engineering of proteins with an extended shelf-life for practical applications of benefit to society. Broader impacts. The project will provide the community with a simple, inexpensive, sensitive, and accessible assay to quantify the kinetic stability of any protein of interest, either purified or in its biological milieu. Furthermore, this project has the potential to provide novel insight about the biological role of protein kinetic stability that could catalyze further studies and advance our theoretical understanding about the role of chemistry and physics in life processes. From an educational perspective, this project will involve postdoctoral, graduate, and undergraduate researchers working together in a stimulating environment that integrates research training and learning. Learning through hands-on research is the most effective method for exposing undergraduate students to science and its career opportunities. Since its inception, the lab of the principal investigator has been engaged in training and mentoring many undergraduate students, including women and underrepresented minorities. Undergraduate students have been coauthors in numerous publications. In addition to research training, students involved in this project will participate in frequent group meetings that will provide a forum to present their research and develop their communication skills. Yearly participation at local or national meetings will give them an opportunity to disseminate their results through poster presentations. Students will be actively involved in writing early drafts of manuscripts for publication in peer-reviewed journals.