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Proximity of proregion anionic residues to the mature region maintains proCryptdin‐4 inhibition
Author(s) -
FIGUEREDO SHAREL MENEZES,
Gable Jonathan E,
Kim Judy E,
Ouellette Andre J
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.521.2
Subject(s) - förster resonance energy transfer , chemistry , mutant , mutagenesis , amino acid , biophysics , biochemistry , n terminus , moiety , stereochemistry , cysteine , peptide sequence , enzyme , fluorescence , biology , gene , physics , quantum mechanics
α‐Defensins are synthesized as inactive precursors that must undergo proteolytic conversion to mature bactericidal peptides. Acidic amino acids near the proCrp4 N terminus mediate inhibition of proCrp4 as shown by selective deletion and charge neutralizing mutagenesis (J. Biol. Chem. 284: 6826–6831, 2009). To study the inhibitory mechanism, we have compared the proximity of those anionic residues to the Crp4 component of native and mutant proCrp4 molecules using Förster resonance energy transfer (FRET). To provide FRET probes and enable FRET conformational studies in native and charge neutralized proCrp4, single Crp4 Trp substitutions were introduced in the α‐defensin moiety at V58W, F66W, and at the C terminus, and a Cys residue was added to proCrp4 N terminus (C N ) for IAEDANS labeling. ProCrp variants had bactericidal activities comparable to the parent molecules and were processed correctly by MMP‐7. FRET efficiency and distances between the FRET pairs in native and charge‐neutralized proCrp4 were determined. The N and C‐terminal regions of inactive C N (V58W)‐proCrp4 are closest with a FRET pair distance of 11.5 Å. In the active, bactericidal C N ‐(DE/G‐V58W)‐proCrp4 mutant, this distance increased to 14.3 Å. Thus, mutations that relieve proCrp4 inhibition do so without markedly repositioning the relation of the proregion N terminus to the Crp4 component of the precursor. Supported by NIH grants DK044632 and AI059346..