CAREER: Thermodynamic and Structural Mechanisms in Protein Fold Adaptation and Thermostability

Claflin University, Orangeburg SC

Investigators

Abstract

Intellectual Merit Understanding how to redesign enzymes to function at different temperatures is critical to increasing the efficiency of many industrial processes including the production of biofuels. It is also crucially important to understanding the basic principles of protein folding, adaptation, and stability. This project tests and applies two novel hypotheses in protein adaptation and stability: 1) that protein adaptation is "Fold-Specific," meaning that the molecular adaptation strategy taken to alter an enzyme's temperature range is dependent on its specific enzymatic fold and 2) that increases in the stability of the native state of a protein can be achieved by limiting the conformations accessible in the unfolded state through steric exclusion of non-native like conformations - a novel mechanism termed 'Entropic Stabilization.' Already, many mutations that have no apparent effect on the folded state structure have been observed to increase thermostability, and the hypotheses stated above are proposed as a fundamental mechanism to explain this phenomenon. In the process of testing these hypotheses, several enzymes (alpha-glucosidases and cellulases) in the most common protein fold, the 8 stranded alpha/beta barrels, will be redesigned to function at the high temperatures consistent with the needs of promising industrial processes. Using high throughput, low error rate, random mutagenesis and chromogenic selection, several enzymes which show increased thermostability will be selected. In order to achieve higher catalytic rates and efficiencies, enzymes from psychrophilic, or "cold-loving," organisms will be used as a starting point. From multiple attempts of this process, a structural map of "hot spots" in the alpha/beta barrel architecture that show a high probability of involvement in temperature adaptation will be generated. These "hot spots" will be compared to those identified from a structural database, developed by this project, of all known alpha/beta barrel structures from mesophilic, thermophilic, and psychrophilic bacteria to posit general, fold specific rules for protein design. In a complimentary approach, those thermostable mutants that exhibit no obvious structural mechanisms for thermostability in the native-state will be compared using hard-sphere steric ab initio molecular modeling to enumerate all possible sterically-allowed conformations for tetra-peptide sequences in the protein. These will be classified as native-like and non-native and will then be compared to similarly calculated conformational space ratios for the structurally equivalent regions of wild-type enzyme and correlated to their propensity for increasing thermostability. In order to classify which conformations are ruled in or out, and which are native-like and non native, a novel 3D Ramachandran Map-like tool that graphically represents different tetrapeptide conformations will be developed. Broader Impact In 2009, Forbes ranked Claflin University as the Best Historically Black University in the nation. Over 90% of the student body is African American and 60% are first generation college students, making Claflin a vital part of the regional and national effort to recruit minority students into advanced degrees in STEM fields. By incorporating this research into four different project-based lab courses at both graduate and undergraduate levels, students will learn traditional techniques such as protein purification, PCR, kinetics measurements, and bio computational analysis, and will apply them in semester-long projects. In order to stimulate enthusiasm, the top-performing students (with the largest number of successful mutants or best computational results) will earn one trip to a national research conference to present their results. Thus, this project encourages over 40 African American and first generation students per semester (all biology and biochemistry majors at Claflin) to participate in and learn about cutting edge bio fuel research. The project will introduce the field to a whole generation of students, while at the same time supporting the development of sustainable energy by unlocking a critical step in biofuel production.