Approximately 60% of the recombinant His 6 -proHNP1 was folded with native disulfide linkages (Fig. 2) and misfolded propeptide was readily converted to the folded form in buffer containing urea and reduced/oxidized glutathione (Fig. S1 ). The expression of folded and misfolded forms of proHNP1 allowed for the analysis of the structural requirements for HNP1-mediated bactericidal activity in vitro. CNBr treatment of folded (proHNP1A) or misfolded (proHNP1B) precursor generated different HNP1 conformers (Fig. 3) . Despite the generation of oxidized peptides with masses matching that of natural HNP1, only the conformer generated by CNBr cleavage of proHNP1A had microbicidal activity against S. aureus. Processing of proHNP1A by NE or PR3 generated HNP1 peptide identical to natural HNP1, but proHNP1B was heterogeneous and was degraded by NE, PR3 and CG. These data demonstrate that the native HNP1 fold is necessary for proteolytic stability and accurate processing of the HNP1 propeptide by the three azurophil granule serine proteases investigated.