Grant: Discovery Grants Program - Individual

Proteins are the doers in cells. They are involved in building, degrading, transporting, importing and exporting cellular components. In order to carry out these functions, proteins must fold correctly into three-dimensional shapes that enable precise interactions with other molecules such as proteins, DNA, RNA, metabolites, metal ions, or combinations of these. In addition, proteins must function only when and where required. For example, some proteins like hemoglobin, which is packed in red blood cells and carries oxygen to our cells, must function for a long time, while other proteins, like those involved in blood clotting, must act quickly but only transiently. Moreover, our cells are not simple bags of molecules, but instead are comprised of subcellular compartments (e.g., the nucleus or mitochondrion or chloroplast) where different processes occur. So, proteins must be sorted to their proper cellular destinations to perform those activities. The proper functioning of any protein results from a combination of four factors: location, activity, structure and stability. To understand how these four factors are entwined, we study one model protein called tRNA nucleotidyltransferase (tRNA-NT). This vital protein is an enzyme that builds a small extension on the end of transfer RNAs (tRNAs) so that amino acids can be attached. These tRNAs with amino acids act as adaptor molecules to translate the genetic code into the sequence of amino acids in a protein. Therefore, without tRNA-NT cells do not make proteins. Because tRNA synthesis and repair occur in multiple places in the cell (nucleus, mitochondrion, cytosol and in plants, chloroplast) tRNA-NT must be directed to these locations. To study tRNA-NT, we first make changes in specific regions of the protein to see how these changes affect location, activity, structure and stability. Some of the changes that we have made affected multiple parameters. For example, we showed that changing certain amino acids in a plant tRNA-NT reduced that variant's thermal stability, eliminated its ability to extend tRNA (reduced activity), and caused a change in intracellular distribution (changed localization). Similarly, we showed that a single amino acid change in the yeast enzyme could reduce its thermal stability and activity. We will use a wide variety of cell and molecular biological approaches as well as biochemical and biophysical techniques to study the interplay among location, activity, structure and stability in tRNA-NT function. We also are identifying proteins that interact with tRNA-NT to ensure proper maintenance of activity and localization in cells. Our studies allow us to develop a better understanding of how these factors combine to ensure protein function. This fundamental knowledge will enable us to optimize delivery of functional proteins in cells.