The α-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance.

Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensincryptdin-4 (Crp4) and peptide variants with mutations at Arg7 or Glu15 residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability.

Expression and purification of recombinant alpha-defensins and alpha-defensin precursors in Escherichia coli.

Recombinant expression of alpha-defensins can be obtained at efficient levels in Escherichia coli. Amplified alpha-defensin or pro-alpha-defensin coding cDNA sequences are cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells.

Inhibition of bactericidal activity is maintained in a mouse alpha-defensin precursor with proregion truncations.

alpha-Defensin biosynthesis requires the proteolytic conversion of inactive precursors to microbicidal forms. In mouse Paneth cell pro-alpha-defensin proCrp4((20-92)), anionic amino acids positioned near the proregion N-terminus inhibit proCrp4 activity by an apparent charge neutralization mechanism. Because most pro-alpha-defensins contain proregions of highly conserved chain length, we tested whether decreasing the distance between the inhibitory acidic residues of the proregion and the alpha-defensin component of the precursor would alter proCrp4 inhibition.

Anionic amino acids near the pro-alpha-defensin N terminus mediate inhibition of bactericidal activity in mouse pro-cryptdin-4.

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption.

Serum protein-expression profiling using the ProteinChip biomarker system.

Protein-expression profiling of serum is a common approach to the discovery of potential diagnostic and therapeutic markers of disease. Like any other proteome, the serum proteome is characterized by protein expression across a large dynamic range. This single facet requires the employment of fractionation procedures prior to detection of protein.