We used a two-stage radial diffusion assay to perform a structure-acti
vity study of the antifungal effects of protegrin-1 (PG-1) on yeast-ph
ase Candida albicans, While doing so, we computed MICs from the radial
diffusion assay data by three methods and compared the respective val
ues with results from colony count and broth microdilution assays. Thi
s allowed us to identify several technical modifications that improved
the sensitivity and accuracy of radial diffusion assays. We found tha
t both PG-l and enantiomeric PG-1 (composed exclusively of D-amino aci
ds) were potently fungicidal for yeast-phase C. albicans, The protegri
ns PG-2, -3, and -5, but not PG-4, were as effective as PG-1, At least
one intramolecular disulfide bond was required to retain optimal cand
idacidal activity at physiological NaCl concentrations. Truncated vari
ants of PG-l that lacked its first four residues showed decreased cand
idacidal activity, although their activity against bacteria,vas substa
ntially intact. Altering the beta-turn region (residues 9 to 12) of PG
-l or its variants further decreased candidacidal activity. These stud
ies suggest that only 12 residues are needed to endow protegrin molecu
les with strong antibacterial activity and that at least 4 additional
residues are needed to add potent antifungal properties. Thus, the 16-
residue protegrin PG-2 likely represents the minimal structure needed
for broad-spectrum antimicrobial activity encompassing bacteria and fu
ngi.