Enkephalin peptide neurotransmitters are endogenous opioid peptide neurotransmitters that are synthesized from proenkephalin (PE) by proteolytic processing. Because the mature processed enkephalin neuropeptide (not PE) is biologically active as a peptide neurotransmitter, participating in pain relief and brain functions, proteolytic processing is crucial for neurotransmitter activities of enkephalins. Recent findings have indicated a key role for secretory vesicle cathepsin L in processing PE to active enkephalin peptides. To understand the PE proteolytic processing mechanism, the goal of this project will be to investigate the conformational features of PE cleavage sites during processing by cathepsin L, combined with defining interacting domains of PE-cathepsin L complexes . This goal will assess the hypothesis that conformational features of PE cleavage sites participate in the proteolytic processing by cathepsin L. This project will be achieved in three specific aims. The first aim will assess the relative accessibilities of PE cleavage sites to the aqueous environment in DXMS studies exposure to aqueous solvent, using recombinant PE in hydrogen deuterium exchange-mass spectrometry (DXMS) studies conducted under pH conditions representing those within secretory vesicles where PE processing occurs. The second aim will define the cathepsin L-mediated cleavage sites of PE and HMW PE-derived intermediates, and compare these cleavage sites with their conformational features from DXMS investigations. The third aim will study PE-cathepsin L interactions by DXMS and direct chemical cross-linking studies to understand conformational features of the complex. This project will have gained knowledge concerning the conformational features of PE cleavage sites with respect to accessibilities to the aqueous environment, PE cleavage products generated by cathepsin L, and interacting domains of PE-cathepsin L complexes. Results will provide new knowledge of the proteolytic mechanisms utilized for biosynthesis of active enkephalin opioid peptide neurotransmitters.