The solution structure of human salivary histatin 5 (D‐S‐H‐A‐K‐R‐H‐H‐G‐Y‐K‐R‐K‐F‐H‐E‐K‐H‐H‐S‐H‐R‐G‐Y) was examined in water (pH 3.8) and dimethyl sulfoxide solutions using 500 MHz homo‐ and heteronuclear two‐dimensional (2D) nmr. The resonance assignment of peptide backbone and side‐chain protons was accomplished by 2D total correlated spectroscopy and nuclear Overhauser effect (NOE) spectroscopy. The high J NH‐C αH values (≥7.4 Hz), absence of any characteristic NH‐NH(i, i + 1) or CαH‐CβH(i, i + 3) NOE connectivities, high dδ/dT values (≥0.004 ppm K−1) and the fast 1H/2H amide exchange suggest that histatin 5 molecules remain unstructured in aqueous solution at pH 3.8. In contrast, histatin 5 prefers largely α‐helical conformation in dimethyl sulfoxide solution as evident from the J NH‐C αH values (≤6.4 Hz), slow 1H/2H exchange, low dδ/dT values (≤0.003 ppm K−1) observed for amide resonances of residues 6–24, and the characteristic NH‐NH(i, i + 1) and CαH‐CβH(i, i +3) NOE connectivities. All backbone amide 15N‐1H connectivities fall within 6 ppm on the 15N scale in the 2D heteronuclear single quantum correlated spectrum, and the restrained structure calculations using DIANA suggest the prevalence of α‐helical conformations stabilized by 19 (5 → 1) intramolecular backbone amide hydrogen bonds in polar aprotic medium such as dimethyl sulfoxide. The interside‐chain hydrogen bonding and salt‐bridge type interactions that normally stabilize the helical structure of linear peptides in aqueous solutions are not observed. Histatin 5, unlike other naturally occurring antimicrobial polypeptides such as magainins, defensins, and tachyplesins, does not adopt amphiphilic structure, precluding its insertion into microbial membranes and formation of ion channels across membranes. Electrostatic (ionic type) and hydrogen bonding interactions of the positively charged and polar residues with the head groups of microbial membranes or with a membrane‐bound receptor could be the initial step involved in the mechanism of antimicrobial activity of histatins. © 1998 John Wiley & Sons, Inc. Biopoly 45: 51–67, 1998

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Bactenecin 5 and its fragments [BN22 (1-22), BN16 (7-22), and BC24 (20-43)] were synthesized by solid-phase methods. Their antifungal activities on Candida albicans have been studied and compared with those of the native bactenecin 5. The conformational preferences of these peptides in aqueous and nonaqueous solutions and in lipid vesicles were examined by circular dichroism. The highly active N-terminal fragment (BN16) was examined in aqueous solution using 500 MHz two-dimensional NMR. Bactenecin 5 and its fragments are potent candidacidal agents against C. albicans. The N-terminal fragments (BN22 and BN16) of bactenecin 5 are relatively more active than the C-terminal fragment BC24, especially at lower concentrations. The N-terminal region (7-22) which retains the activity of the whole molecule appears to be the functional domain for candidacidal activity. The CD spectra of bactenecin 5 and its fragments are reminiscent of the CD spectrum of poly(L-proline) type II structure in aqueous and nonaqueous solutions and also in lipid vesicles. The temperature dependence of NH chemical shifts and 1H/2H exchange effect on amide resonances suggest the absence of intramolecularly hydrogen-bonded NH groups. The coupling constant (JNH-CalphaH) values, conformational restriction offered by the Pro residues (phi = -60 degrees +/- 15 degrees), the set of medium- and short-range nuclear Overhauser effects observed for the active N-terminal fragment (BN16), and the restrained structure calculation using DIANA suggest that poly(L-proline) type II conformers of the peptide molecules could be significantly populated in aqueous solution. The ability of bactenecin peptides to induce disruption of lipid vesicles correlates well with their activity. Our results suggest that poly(L-proline) type II structure may, indeed, be the biologically active conformation for candidacidal activity of bactenecin peptides.

The 'fuzzy end elimination theorem' (FEE) is a mathematically proven theorem that identifies rotameric states in proteins which are incompatible with the global minimum energy conformation. While implementing the FEE we noticed two different aspects that directly affected the final results at convergence. First, the identification of a single dead-ending rotameric state can trigger a 'domino effect' that initiates the identification of additional rotameric states which become dead-ending. A recursive check for dead-ending rotameric states is therefore necessary every time a dead-ending rotameric state is identified. It is shown that, if the recursive check is omitted, it is possible to miss the identification of some dead-ending rotameric states causing a premature termination of the elimination process. Second, we examined the effects of removing dead-ending rotameric states from further considerations at different moments of time. Two different methods of rotameric state removal were examined for an order dependence. In one case, each rotamer found to be incompatible with the global minimum energy conformation was removed immediately following its identification. In the other, dead-ending rotamers were marked for deletion but retained during the search, so that they influenced the evaluation of other rotameric states. When the search was completed, all marked rotamers were removed simultaneously. In addition, to expand further the usefulness of the FEE, a novel method is presented that allows for further reduction in the remaining set of conformations at the FEE convergence. In this method, called a tree-based search, each dead-ending pair of rotamers which does not lead to the direct removal of either rotameric state is used to reduce significantly the number of remaining conformations. In the future this method can also be expanded to triplet and quadruplet sets of rotameric states. We tested our implementation of the FEE by exhaustively searching ten protein segments and found that the FEE identified the global minimum every time. For each segment, the global minimum was exhaustively searched in two different environments: (i) the segments were extracted from the protein and exhaustively searched in the absence of the surrounding residues; (ii) the segments were exhaustively searched in the presence of the remaining residues fixed at crystal structure conformations. We also evaluated the performance of the method for accurately predicting side chain conformations. We examined the influence of factors such as type and accuracy of backbone template used, and the restrictions imposed by the choice of potential function, parameterization and rotamer database. Conclusions are drawn on these results and future prospects are given.

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