Proteins perform a particularly diverse range of tasks in biological systems. To study how these molecules function in vitro and in vivo and to create new molecular entities with therapeutic and diagnostic capabilities, researchers have developed a broad range of methods to chemically modify proteins. While all of these methods are useful, they also all have disadvantages that limit their utility. Recently, we developed a method for enzymatically modifying proteins using the enzyme protein farnesyltransferase (PFTase). This method allows azide and alkyne-containing substrates to be transferred to proteins containing a small, four residue, and sequence at their C-terminus. Subsequent bio- orthogonal reaction of the azide or alkyne-functionalized protein can be used to prepare a wide variety of protein conjugates. What makes this approach unique is the fact that it allows selective covalent protein modification to be achieved with a minimalist tag. In this application we propose to capitalize on the utility of this protein modification strategy by applying it to several important problems in therapeutics while we continue to improve and refine it. The specific aims of this project are: (1) Design and synthesize simplified azide-, alkyne-, and other functional group-containing substrates for PFTase (2) Develop a general directed evolution system to produce useful new mutants of PFTase including enzymes with relaxed and/or alternative isoprenoid and peptide/protein substrate specificity. (3) Use the PFTase catalyzed enzymatic protein modification method to prepare PEGylated forms of erythropoietin (EPO) and brain derived neurotrophic factor (BDNF) with increased stability and evaluate their neuroprotective efficacy following intranasal application in a rat hypoxia model. (4) Use a similar approach to prepare antibody-RNA conjugates that can be used to target siRNAs to T cells. If successful, the work described in this application could lead to improved protein- based drugs that could be used for the treatment of stroke, Alzheimer's and other neurodegenerative conditions as well as new, more selective agents for the treatment of autoimmune diseases and T cell leukemias. Moreover, the protein modification methodology developed here should be useful for the preparation of a wide variety of other protein conjugates that could be employed for a plethora of therapeutic and diagnostic applications.