Fluorescence stereo microscopy for exploring the three-dimensional dynamics of receptor ligand interactions

1 This MIRA project will advance understanding of the function of tyrosine sulfation, a fundamental 2 modification that regulates diverse biological functions. For example, sulfation plays a critical role 3 in entry of HIV into human cells, in the interaction of microbial molecules with host immune 4 receptors and in the activity of tick-derived peptides, which hold potential for treatment of blood

5 clotting associated with cardiovascular diseases. Despite the increasing awareness of the 6 importance of tyrosine sulfation, most sulfated peptide complexes have not yet been fully 7 characterized. Further, we have only a partial view of the components essential for transducing 8 the response of such activated complexes. We also have limited strategies for efficiently

9 producing and testing the therapeutic potential of sulfated molecules. The PI is well positioned to 10 address these challenges. With support from the NIH, the PI discovered the host XA21 immune 11 receptor, a protein that is representative of a large class of plant and animal receptors. Further, 12 the PI’s team isolated and characterized a sulfated peptide secreted by a Gram-negative

13 bacterium that binds to the XA21 receptor and activates the immune response. We propose 14 research in three related areas: We will isolate and characterize the diversity of receptors that 15 bind sTyr peptides, identify and analyze sTyr binding interfaces and pioneer a strategy to produce 16 novel sulfated molecules in an efficient manner. To accomplish our goals, we will employ new

17 experimental tools and approaches. These include use of established whole genome sequenced 18 mutant lines to rapidly assess phenotypes of newly discovered genes, sensitive assays to assess 19 ligand binding, state-of-the-art mass yeast cell surface display and spectrometry approaches to 20 engineer receptors and identify key components of immune complexes. The knowledge gained

21 from this research will provide new insights into an essential biological process, laying the 22 foundation for the development of reagents capable of inhibiting or activating cellular interactions 23 with a high degree of affinity and specificity with potential applications in research, agriculture and

24 medicine.