title: Conformational landscape and pathway of disulfide bond reduction of human alpha defensin.
abstract: 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.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CH Utrecht, The Netherlands.|Netherlands Proteomics Centre, 3584, CH Utrecht, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Snijder
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CH Utrecht, The Netherlands.|Netherlands Proteomics Centre, 3584, CH Utrecht, The Netherlands.
authors.forename: Michiel
authors.initials: M
authors.lastname: van de Waterbeemd
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Glover
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana.
authors.forename: Liuqing
authors.initials: L
authors.lastname: Shi
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana.
authors.forename: David E
authors.initials: DE
authors.lastname: Clemmer
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CH Utrecht, The Netherlands.|Netherlands Proteomics Centre, 3584, CH Utrecht, The Netherlands.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D058767:Protein Unfolding; D023084:alpha-Defensins / Q000737:chemistry*
chemical_list: C500225:DEFA5 protein, human; D004220:Disulfides; D023084:alpha-Defensins; D003545:Cysteine
keywords: antimicrobial peptide; defensin; disulfide bond; ion mobility spectrometry; mass spectrometry; reductive unfolding; tandem MS