Protein acylation is a powerful post-translational signaling mechanism. In contrast to the transcriptional N-myristoylation class of acyl proteins, pure proteins have not been available to study the other class of acyl proteins where internal residues are post-translationally fatty acylated. The applicant's long term objective is to study the biochemistry of protein internal acylation as a feature of signal transduction in mammalian proteins such as Ras. The proposed research is a unique opportunity to study, using purified proteins, the enzymology and biochemistry of an internal acylation of a protein which causes a profound change in its function. A bacterial toxin system is used as a model of protein internal acylation. Hemolysin (HlyA) typifies the RTX toxins, a family of cytotoxic proteins secreted by different genera of Gram negative bacteria which bind and lyse specific mammalian cells. The toxins are remarkable for their unusual activation, mode of secretion, glycine-rich nonapeptide repeats (RTX), and different cell specificities. HlyA originates as nontoxic proHlyA which is post-translationally modified to toxic HlyA by long chain fatty acylation catalyzed by HlyC, a transacylase. Acyl-ACP is reportedly the obligatory acyl donor for the internal fatty acylation. Acylation is not essential for secretion, but acylation is the single factor that renders the protein toxic. Using different recombinant DNA vectors, HlyC and proHlyA have been over produced. The activation of proHlyA to HlyA catalyzed by HlyC will be studied with a variety of acyl-ACPs which have been synthesized with different radioactive or fluorescent acyl groups in order to define the reaction optimum conditions, preferred substrate, stoichiometry, kinetics and mechanism. proHylA + *acyl-SACP---*HLYA + ACPSH. The purified transacylase, HlyC, will be characterized. Changes in HlyA characteristics upon activation will be examined by Fourier transform infrared spectroscopy (conformational changes) and hydrodynamic light scattering and fluorescence anistropy (change in aggregation tendencies). Several homologous systems with different biological functions and target cell specificities use the hemolysin scheme of generation of toxicity and protein secretion. Examples range from the secreted rhizobial bacterial protein NodO (homologous to HlyA) that infects legumes causing nodulation to HlyA itself which is epidemiologically important in humans. The hemolysin scheme is a recurring biological motif of rendering a protein toxic and secreting the infectious, cellular specific protein. This motif is used to infect human, plant, and animals cells en route to achieving different objectives.
