Site of fluorescent label modifies interaction of melittin with live cells and model membranes

Abstract

The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

Investigation of the structure‐activity relationship in ponericin L1 from Neoponera goeldii

“…[ 15‐17 ] These synthetically accessible systems are also amenable to incorporation of fluorescent or other spectroscopic probes as well as nonnatural amino acids, further expanding the utility as a biophysical model system. [ 14,18‐21 ] Among these, by far, melittin from Apis mellifera is the most widely studied and well understood with several high resolution structures solved in both solution and bound forms [ 22,23 ] as well as numerous molecular dynamics simulations of the peptide interaction with bilayers. [ 15,24‐26 ]…”

C‐Terminal Modification and Multimerization Increase the Efficacy of a Proline‐Rich Antimicrobial Peptide

“…Importantly, these linkers have been demonstrated to possess inherent fluorescent properties that enable their use in, for example, high resolution microscopy. Such inbuilt fluorescence would be an advantage in tetrameric PrAMPs, as we have previously shown that addition of a bulky AlexFluor moiety to our first tetramer resulted in significant loss of antimicrobial activity . Consequently, we undertook the use of dibromomaleimide conjugation of Chex1‐Arg20 dimers possessing various C‐terminal chemical modifications to form non‐disulfide‐linked tetramers with this inbuilt‐fluorescent capability.…”

“…[13] Importantly,t hese linkers have been demonstrated to possess inherent fluorescent properties [13c, 14] that enable their use in, for example, high resolution microscopy.S uch inbuilt fluorescencew ould be an advantage in tetrameric PrAMPs, as we have previously shown that addition of ab ulky AlexFluor moiety to our first tetramer resultedi ns ignificant loss of antimicrobial activity. [8,15] Consequently,w eu ndertook the use of dibromomaleimide conjugation of Chex1-Arg20 dimers possessing various C-terminal chemicalm odificationst of orm non-disulfide-linked tetramers with this inbuilt-fluorescentc apability.Asecond series of tetrameric analogues was also prepared using dimeric peptides linked by trans-1,4-dibromo-2-butene, a,a'-dibromo-p-xylene or 6-bismaleimidohexane as tether to determine by diffusion NMR the effect of tetherl ength on antimicrobial activity.…”

Phosphorylation of a full length amyloid-β peptide modulates its amyloid aggregation, cell binding and neurotoxic properties

“…These Ab42(S26C) and pAb42(S26C) peptides were synthesized and labelled with AlexaFluor 430 as NHS ester forms (Invitrogen, Sydney, Australia) using thiolmaleimide conjugation chemistry. 14 The coupling reaction between NHS-AlexaFluor 430 (1.4 mmol, 1 mg) and N-(2-aminoethyl) maleimide (1.68 mmol, 0.43 mg) was performed in the presence of N,N-diisopropylethylamine (2.68 mmol, 0.05 ml) in dimethylformamide. Mal-AlexaFluor was used to react with the side chain of the cysteine residue.…”

“…The Ab42 and pAb42 peptides were synthesized using previously reported in-house procedures for peptide synthesis, 14 but a number of modifications and optimisation steps were required to achieve high levels of purity and yields. The peptides were synthesised using the Fmoc/tBu solid phase strategy with microwave irradiation at 86 1C on a CEM Liberty synthesizer.…”