The principles of evolution are well known and are reflected in the beauty and complexity of life on this planet. Understanding how life evolves and changes through time is an important question in modern biology. Even before DNA was discovered to be the hereditary material of life, the ideas underlying evolution could be observed readily by the domestication of plants and animals. Perhaps one of the most well known examples of domestication is the dog, once a wild wolf and now a companion. Though it is easy to appreciate the outward differences between a dog and a wolf, it is more difficult to determine how small changes in the genes and the proteins encoded by them give rise to changes in the characteristics of each breed. The PI studies the evolution of molecules and how changes in environmental conditions such as temperature select for changes (mutations) that make those molecules better adapted. Typically, the adaptation of organisms to new conditions takes much longer than is practicable in lab studies. By focusing on a simple thermophilic organism (a bacterium that lives at high temperatures) that reproduces in 30 minutes, thousands of generations can be studied during a single laboratory experiment. To facilitate these studies the ''weak link'' approach was developed with previous NSF support. In the ''weak link'' approach, survival of a genetically modified thermophilic organism (Geobacillus stearothermophilus) is dependent on changes to an essential molecule that only functions at lower temperature. In order to reproduce at higher temperature, mutations must occur to the essential gene. By growing the bacteria at low temperature and then gradually increasing the temperature the bacteria can be quickly adapted into one that can live at high temperatures. It is then possible to determine how changes to the essential gene produced a protein that was well adapted to work at high temperature and permit the bacteria to survive in the new environment. Experimental evolution of a single gene also provides an opportunity to investigate the functional intermediates of protein adaptive evolution during the course of natural selection. Several fundamental questions can be addressed through this work. For example: How reproducible is evolution? If the experiment is performed three times do the same changes occur and in the same order? What happens if the conditions of the selection change? How will adaptation change the molecule in response? In addition to exploring the basic principles for all life, these findings have application to protein engineering and biotechnology for the production of more rugged proteins.
Broader Impact The integration of science education and research is a primary focus of the investigator''s laboratory. Nine undergraduate and three graduate researchers have carried out work on this project as independent research and have presented findings at scientific conferences and in published research. The breadth of the project from organism to electron density in high resolution structures requires students to become broadly educated in evolution, microbiology, biochemistry and biophysics. The PI is faculty mentor for the NSF-IGERT Program for Cellular Engineering, Houston Molecular Biophysics Program and the Gulf Coast Consortia and is a co-founder of the Rice Center for Evolution.
The PI teaches Biochemistry and incorporates the lessons learned from these experiments into the discussion of protein folding and stability as well as how evolution impacts our world through the rise of drug resistant pathogens. In the last three years, the lab has hosted 7 High School groups (including a High School AP teachers program) as well as mentoring summer researchers from the under represented Hispanic community through the Rice AGEP program. The PI has also used this project as the basis for community based seminars on the nature of evolution (''Evolution in a coffee pot'').
The physical principles that allow an organism to adapt to changes in its environment has important implications for biotechnology and biomedical research. In Nature, evolution occurs through the continuous adaptation of a population to its environment. The success or failure of organisms during adaptation is based upon changes in molecular structure that give rise to changes in fitness that dictate evolutionary fates within a population. While the conceptual link between genotype, phenotype and fitness is clear, the ability to relate these complex adaptive landscapes in a quantitative manner remains difficult. We have created a system where the reproductive success of an organism is dependent on the function of a single gene, which, in our case, ceases its essential function at high temperatures. We used this ‘weak link’ method to favor mutations to the maladapted adenylate kinase gene within a microbial population that resulted in the identification of mutations to this gene that arose nearly simultaneously and competed for success. The unique catalytic role of adenylate kinase in vivo is to maintain adenylate homeostasis and thus retain a high concentration of the energy carrier ATP, which is used to promote various vital reactions within the cellular milieu. Studies showed the necessary mutations conserved the unique function while extending it to higher temperatures through combinations of increased activity and stability. In vitro and in vivo activities are restricted by the same principles. Like in vivo activity, in vitro enzyme activity is a product of critical catalytic and folding pathways, and hence is a valuable proxy for fitness. The fitness function developed in our study directly links the unique properties of an enzyme to evolutionary fates in a quantitative and predictive manner through a comparative study of empirical and simulated data. The success or failure of organisms during evolution is dictated by changes in molecular structure that give rise to changes in fitness revealed by evolutionary dynamics within a population. We have shown that predicting success during adaptation can depend critically upon enzyme kinetic and folding models. In addition we have used biophysical methods such as X-ray crystallography to elucidate the physical basis for how mutations change adenylate kinase structure and function to accommodate the adaptive changes required by selection. The fitness function thereby links organismal adaptation to the properties of a single gene. Understanding the physical basis for adaptation of an organism is the first step in the development of approaches that can accurately model, and someday predict, the manner in which organisms would respond to new antibiotics and improve upon the current clinical regimens. Just as importantly, this proposal was used to train three graduate Ph.D. students and six undergraduate researchers in this highly interdisciplinary and integrative application of biophysics to evolutionary biology. This is a relatively new area that holds great promise for engineering or evolving proteins to specific needs in biotechnology as well as in basic discovery science.
