In order to better understand which atoms in a protein contribute decisively to the key properties that support enzyme function, this project will generate libraries of natural proteins that contain different classes of mutations (random substitutions, glycine substitutions, backbone fission, circular permutation, insertions, and deletions) and examine how each mutation type affects protein folding and function within cells. Adenylate kinases will be used for these studies, since an abundance of pre-existing biophysical data can be tapped to interpret results from systematic mutagenesis experiments. This research will establish if the pattern of a protein's functional tolerance to different classes of mutation contains information on how different native positions within its primary sequence contribute to structure, stability, folding, dynamics, and function. This experimental strategy will be potentially useful for studying any protein for which an in vivo screen is available, including poorly behaved proteins that cannot readily be examined by in vitro physical methods.
Broader Impact This broader impact of this project will be to provide training for graduate and undergraduate students interested in science careers. The PI and graduate students performing this research will conduct annual round table discussions for undergraduates enrolled at a nearby community college. These community college students begin their education in lecture courses and are not necessarily exposed to an environment that mirrors a research lab. Round table discussions will be used to increase student self-confidence by showing beginning college students that they can read a peer-reviewed research article, demonstrate to these students the benefits of research training, and facilitate community college student recruitment to research experiences. These efforts will vertically integrate undergraduate and graduate education by providing beneficial training for graduate students, building self-sustaining outreach from a research university to a minority serving community college, and offering research experiences for community college students.
