This Major Research Instrumentation award funds the acquisition of an automated small cell isothermal titration microcalorimeter for the Emory University Department of Biochemistry's "Macromolecular Structure and Interactions Suite." A long-term goal for the assembled team of investigators is to understand the biochemical basis for macromolecular interactions using a combination of structural biology and biophysical techniques. The new microcalorimeter enhances Emory's diverse research programs that include the investigations of protein synthesis regulation by the ribosome; protein, DNA and RNA modifications in epigenetics, protein sorting, protein degradation and antibiotic resistance; lipid transport; and nuclear receptor function. Understanding the biophysical parameters that govern macromolecular interactions adds a complementary dimension to Emory University structural biology programs. The new equipment therefore enhances training in biophysical experimental methods by improving access to state-of-the-art instrumentation for post-doctoral fellows, graduate students and undergraduates. Furthermore, high school students and teachers will get hands-on experience in a joint Emory-Georgia Tech summer program designed to study the biophysical characterization of extremophile ribosomes. The results of these research and teaching efforts will be broadly disseminated through abstracts and peer reviewed publications, as well as by active participation of students and faculty at professional meetings.
Biological molecules such as proteins, nucleic acids (DNA and RNA), small molecules and other cellular components must interact with each other to perform their biological functions. Therefore, in order to understand the basic mechanisms at the heart of biology, we need ways to examine these molecular interactions in the finest detail. Similarly, the development of new drugs or diagnostic tools is dependent upon a detailed understanding of molecular interactions. Various broadly applicable experimental approaches to measure molecular interactions are available but many suffer from specific limitations, including but not limited to, the requirement for specific alteration of one molecule with a radioactive or non-natural label, or the immobilization of one or both components on a surface or membrane. The technique of "isothermal titration calorimetry" (or ITC) is considered a gold standard for the measurement of molecular interactions in solution and without need for labels or immobilization. In the past, a major limitation of ITC was the relatively large amounts required of each molecule (often costly or difficult to prepare, or both), placing it beyond the scope of many potential applications. Additionally, the complexity and sensitive nature of the equipment limited its use to expert users, not suited to being a shared instrument open to many users of varying experience. Recently, developments in ITC technology have allowed miniaturization of the instrumentation (with an associated reduction in sample requirement) and full automation of sample handling, instrument set-up and experiment execution. These developments have produced instruments that make ITC feasible for new areas of research and by relatively novice experimenters after minimum training (typically only a few hours of instruction and initial supervised used). This award funded the purchase of new shared instrument, an "Auto-iTC200" fully automated small volume microcalorimeter manufactured by MicroCal (part of GE Healthcare). The instrument was installed in December 2010 and continues to be located in a shared equipment laboratory within Emory University’s Department of Biochemistry. Initial training for 35 users was provided with the purchase of the instrument and took the form of two full day visits by MicroCal/ GE specialists. The first covered basic ITC theory and instrument use and care, and the second, specialist applications and data analysis. Subsequent training for new users was provided directly by the award principle investigator (Dr. Graeme L. Conn). Over the course of this award more than 50 users have received training and most have used the Auto-iTC200 instrument to advance their research goals. These individuals have included postdoctoral researchers, graduate students, technical staff and several undergraduate summer students. Commitment by the Department of Biochemistry to pay 50% of an annual service contract for the Auto-iTC200 (the remainder coming from use-based fee from user groups) ensures that the instrument will continue as an invaluable shared resource for years to come for researchers within the Emory community, and in the Atlanta area and beyond. The Auto-iTC200 instrument is very broadly applicable to the study of molecular interactions and our instrument has allowed researchers to make major advances in many very different research programs. The following examples are provided to give a flavor of the research being performed and the specific impact of the availability of instrumentation provided by this award. The Conn laboratory took advantage of the high throughput capability of the Auto-iTC200 instrument to quickly develop a deeper understanding of the activity of various RNA modifying enzymes that confer resistance to antibiotics. A recent notable among was the work of an undergraduate researcher, Jennifer Hernandez, who in the summer of 2013, analyzed the contributions of specific amino acids to cofactor and substrate binding by the enzyme Tsr (which confers resistance to thiostrepton, an antibiotic used in veterinary practice). Jennifer will present the results of her research at the 2013 Annual Biomedical Research Conference for Minority Students (ABRCAMS). The Cheng laboratory have used the Auto-iTC200 to study the interactions of protein and DNA modifying enzymes with their coenzymes and substrates, and their roles in epigenetic modification. Prof. Haian Fu’s group used the instrument extensively to measure the interactions of 14-3-3 proteins, conserved regulatory proteins expressed in all eukaryotic cells, and inhibitors. This allowed them to determine binding sites and propose a mechanism for inhibitor action. Other labs have used the instrument to further studies of proteins involved in bacterial survival and resistance to antibiotics, eukaryotic DNA-transcription factor interaction and regulatory mechanisms of proteins that control cell mobility. Results obtained using the Auto-iTC200 have supported successful research grant applications and made important contributions to research publications. The availability of the Auto-iTC200 continues to be advertised to the Emory community and we expect to engage new users with diverse research programs. This instrument has become an invaluable tool for many research groups at Emory; we expect the impact of its availability, made possible by this award, will continue to increase in the future.
