Abstract
The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides.
Highlights
In recent years, a number of natural and synthetic peptides have been investigated for their antimicrobial activity in order to select candidate molecules able to overcome microbial resistance to currently available drugs
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Phosphatidylserine externalisation and reactivity with annexin V was determined by flow cScyyCtt5oo3mmP1e4hettorrwsyyphiihnonaloeotirrdcddeyeellrrlsstet.ooriUanassnessdeeesesxssrtieitffhrtntehhaeeelixpsppaeetpepirottiiinmddeeeassnn,tadaattltrthcheoeaeincirrtdiEEvitCCiito5y50n0 vsvwaaailltduuhoeesspa,tnccenoodeuu,xllnddinoiinnnVedduuowccfeeatshaapepdoopeppteettpoortmssiiidssineiinsnedpCCr..boaayvllbbeiifdcclaaonntwoss iSnCd5u3c1e4awpohpotloescise.llIsn. fUacntd, enrotshteatiesxtpicearlilmy esingtnalificcoanndtidtiioffnesreandcoepwteads,fnouonned obfettwheeepnetphteidpeesrcpernotvaegde otof ainpdoupctoetaicpcoepltlos sinis.thIne faabcste, nncoes(tcaotnisttriocla)lloyrsiingtnhifeicparnetsednifcfeeroefnpceepwtiadsesfo(uFingdurbeet9w).een the percentage of apoptotic cells in the absence or in the presence of peptides (Figure 9)
Summary
A number of natural and synthetic peptides have been investigated for their antimicrobial activity in order to select candidate molecules able to overcome microbial resistance to currently available drugs. The data indicated that the peptide is characterised by a regular decrease of PPII conformational propensi1t0y0 from the N terminus, where the Cys residue is located, to the C-terminus, where Thr and Phe are located (Figure 4). This trend is consistent with previous studies pointing to aromatic and bulky, rigid and hydrophobic side chains as being responsible for a destabilisation of this structural motif [8,9]. After 5 min of treatment the peptide localised on the yeast cell wall (panel A). Phosphatidylserine externalisation and reactivity with annexin V was determined by flow cScyyCtt5oo3mmP1e4hettorrwsyyphiihnonaloeotirrdcddeyeellrrlsstet.ooriUanassnessdeeesesxssrtieitffhrtntehhaeeelixpsppaeetpepirottiiinmddeeeassnn,,tadaattltrthcheoeaeincirrtdiEEvitCCiito5y50n0 vsvwaaailltduuhoeesspa,,tnccenoodeuu,xllnddinoiinnnVedduuowccfeeatshaapepdoopeppteettpoortmssiiidssineiinsnedpCCr..boaayvllbbeiifdcclaaonntwoss iSnCd5u3c1e4awpohpotloescise.llIsn. fUacntd, enrotshteatiesxtpicearlilmy esingtnalificcoanndtidtiioffnesreandcoepwteads,fnouonned obfettwheeepnetphteidpeesrcpernotvaegde otof ainpdoupctoetaicpcoepltlos sinis.thIne faabcste, nncoes(tcaotnisttriocla)lloyrsiingtnhifeicparnetsednifcfeeroefnpceepwtiadsesfo(uFingdurbeet9w).een the percentage of apoptotic cells in the absence (control) or in the presence of peptides (Figure 9)
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