MEMBRANE-INDUCED HELICAL CONFORMATION OF AN ACTIVE CANDIDACIDAL FRAGMENT OF SALIVARY HISTATINS

The conformational preference of the candidacidal C-terminal 16 residu e fragment (9-24; G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) of salivary histati n 5 was examined in water, methanol, and dimethyl sulfoxide solutions using 500 MHz two-dimensional-NMR. Fourier transform infrared and CD s pectroscopy were used to delineate its membrane-bound conformation in lipid vesicles. The peptide backbone and side-chain proton resonance a ssignments were accomplished by two-dimensional total correlated and n uclear Overhauser effect (NOE) spectra. The coupling constant (J(NH-C) alpha(H)) values determined from the double quantum-filtered correlate d spectra, temperature coefficients of NH chemical shifts (ddelta/dT), H-1/H-2 exchange rates on amide resonances, and the set of NOE connect ivities were used to delineate backbone conformational features. The h igh J(NH-C)alpha(H) values (greater-than-or-equal-to 7.4 Hz), absence of any characteristic NH-NH (i, i+1) or C(alpha)H-C(beta)H (i, i+3) NO E connectivities, high ddelta/dT values (greater-than-or-equal-to 0.00 4), and the fast H-1/H-2 amide exchange suggest that the histatin pept ide favors unfolded random conformations in aqueous solution at pH 3.8 . In contrast, the J(NH-C)alpha(H) values (less-than-or-equal-to 6.5 H z), slow H-1/H-2 exchange, low ddelta/dT values (less-than-or-equal-to 0.003) observed for amide resonances of residues 5-16, and the charac teristic NH-NH (i, i+1), C(alpha)H-C(beta)H (i, i+3) NOE connectivitie s, provide evidence for the presence of largely alpha-helical conforma tions in dimethyl sulfoxide, which mimics the polar aprotic membrane e nvironment. In methanolic solutions, 3(10)-helical conformations could exist as a minor population together with the major alpha-helical con formations. Fourier transform infrared spectroscopy and CD data indica te that lipid environments such as dimyristoylphosphatidylcholine vesi cles could induce the peptide to fold into predominantly alpha-helical conformation. The results suggest that in dimethyl sulfoxide and dimy ristoylphosphatidylcholine vesicles the candidacidal domain of salivar y histatin 5 prefers a largely helical conformation, which could facil itate its interaction with the membrane of Candida albicans. The mecha nism of antimicrobial action of this class of polypeptides appears to involve primarily electrostatic and hydrogen-bonding interaction of ca tionic and polar residues with the head groups of the plasma membranes of target cells.