Human alpha defensins are a class of antimicrobial peptides with additional 
antiviral activity. Such antimicrobial peptides constitute a major part of 
mammalian innate immunity. Alpha defensins contain six cysteines, which form 
three well defined disulfide bridges under oxidizing conditions. Residues 
C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the 
peptide. The major tissue in which HD5 is expressed is the crypt of the small 
intestine, an anaerobic niche that should allow for substantial pools of both 
oxidized and (partly) reduced HD5. We used ion mobility coupled to mass 
spectrometry to track the structural changes in HD5 upon disulfide bond 
reduction. We found evidence of stepwise unfolding of HD5 with sequential 
reduction of the three disulfide bonds. Alkylation of free cysteines followed by 
tandem mass spectrometry of the corresponding partially reduced states revealed 
a dominant pathway of reductive unfolding. The majority of HD5 unfolds by 
initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional 
evidence for a minor pathway that starts with reduction of C3-C31, followed by 
C5-C20 and C10-C30. Our results provide insight into the pathway and 
conformational landscape of disulfide bond reduction in HD5.