Our results further support the view that multivalency and oligomerization enhance the uptake without modifying the mechanism of internalization. It has been reported that positively charged peptides are able to induce membrane negative curvature which results in the formation of invaginations, while polymers produce positive curvature resulting in budding In our case, we observed formation of protuberances positive curvature which do not produce budding but seem to be reabsorbed by the GUVs which become larger and slightly loose circularity and roundness.
Moreover, depletion of cholesterol increases polyarginine R8 uptake independently from endocytosis 52 possibly because of a transition from the liquid ordered to the liquid disordered arrangements which favours the interaction. In our case we believe that the further membrane rigidification, caused by the mainly hydrophobic peptides and showed by the calculation of membrane fluidity, could lead to lateral heterogeneity with regions of low tension in between different domains that may be exploited by the peptide to more easily perturb the membrane as also reported by others 35 , 53 , Our study also covers the impact of the structural flexibility on the interaction of peptides with the membrane, as it was shown for several CPPs and related antimicrobial peptides AMPs.
We have shown that oligomerization improves the insertion of gH into a lipid bilayer. The self-association oligomerization plays a key role in the process of membrane interaction and this has also been observed for antimicrobial peptides for which dimerization is known to control selectivity for specific targets and was previously proved also for other CPPs It is interesting to note that this result is in contrast with previous studies on TAT peptide, for which dimerization turned out to have no or little effect on translocation through cellular or model membranes 56 , 57 , 58 , and in fact, TAT is known to essentially employ the endocytic pathway.
The initial steps in peptide membrane interactions is induction of a higher degree of folding into a secondary structure; while surface bound, the peptide inserts into the lipid bilayer and the thermodynamics of this process seem to determine the actual uptake mechanism. Increasing evidences also indicate that membrane interacting peptides may exhibit cross-functionality.
In fact, some AMP possess the ability to cross mammalian cell membranes by non-damaging processes, while several CPPs display significant antimicrobial activity 59 , 60 , 61 , 62 , A high degree of oligomer could be responsible of antibacterial activity while a lower degree could be necessary for membrane crossing. The main difference between multivalency and aggregation may be responsible for the different activities of membranotropic peptides which look apparently similar.
Moreover, we observe an absence of leakage, and hence pore formation, both in the LUVs and GUVs, which seems to be responsible of the lower toxicity of gH in comparison to other CPPs. The H7 moiety does not exert much influence on CPP interaction with a lipid membrane and it does not interfere with pore formation.
Peptides able both to internalize into mammalian cell and kill bacterial pathogens might well act as potential candidates for establishing a novel treatment modality for intracellular infections. Further structural optimization might allow for future development of membranotropic peptides with antimicrobial activity and able to target internalized bacteria 5. In conclusion, multivalency is key for any design strategy which aims at targeting viruses, toxins, proteins, antibodies, and cell surfaces, as many of these biological targets have multiple repetitive binding sites.
Multivalency stands out as a key principle for a targeted strengthening of any interaction between different interfaces correlated to the dramatically enhanced binding on a molecular scale. There are still a few reports analysing this subject in detail, which make a systematic study on models and complex systems and produce reliable predictions for the next chemical step.
Multivalency is clearly advantageous because of its enormous binding strength and is increasingly being applied for pharmaceutically relevant targets 11 , The first successful studies on multivalent drugs have begun to appear. However, this is a new drug concept which focusses on multivalency and holds a great potential in health applications.
Peptides were prepared using the Fmoc-based solid-phase method as previously reported 6 , using a rink amide MBHA 0. Following deprotection, peptides were precipitated in cold ethylic ether and analysis of the crudes was performed by LC—MS using a gradient of acetonitrile 0. Peptide oxidation was performed in ammonium bicarbonate 0. LUVs with a mean diameter of 0. Lipid concentrations of liposome suspensions were determined by phosphate analysis The external buffer was removed by ultracentrifugation at All fluorescence spectra were corrected for the baseline signal.
The excitation generalized polarization GP was calculated as. In particular, labelled and unlabelled vesicles were mixed with a ratio final lipid concentration 0. All results are an average of at least three experiments. Background fluorescence values from solvent and dilution factor were corrected. The association of peptides to lipid bilayers was determined experimentally as a partition equilibrium:. The equilibrium concentration of free peptide in solution, C f , and the extent of peptide binding X b were obtained from f b. A straight line with the slope corresponding to K p is the characteristic of simple partition equilibrium; however, deviations towards increased binding at higher peptide concentrations, are typical for cooperative binding of peptides able to self-associate at the membrane surface.
In the latter case, the surface partition coefficient, K p , is estimated from the initial slopes of the curves. Fluorescence quenching experiments were performed by adding acrylamide. The quenching constants K sv , a measure of the accessibility of tryptophan to acrylamide, were obtained from the slope of the Stern-Volmer equation 74 ; the equation is. As acrylamide does not significantly partition into the membrane bilayer 73 , K sv is a good evidence of the bimolecular rate constant for collisional quenching of the tryptophan residue in the aqueous phase.
Clearly, both the amount of non-vesicle-associated free peptide as well as the fraction of the peptide located at the bilayer surface contributes to K sv. This experiment was conducted in LUVs final phospholipid concentration of 0. Fluorescence was measured before and after the addition of peptides.
In particular, we tested the following peptide concentrations 0. Aggregation A was determined according to the following equation. To get insight into the influence of the peptides on LUVs integrity and size, LUV solution was analysed by DLS; the measurements were also conducted immediately after the addition of increasing concentrations of peptide. We used a BIAcore analytical system Biacore, Uppsala, Sweden equipped with a L1 sensor chip which contains hydrophobic alkanethiol chains, with exposed polar head groups; a lipid bilayer was created by introducing liposomes to the chip.
The plot at each peptide concentration of the SPR angle against time yields a series of sensorgrams which were fitted using numerical integration analysis BIAevaluation software The best fit was obtained by simultaneously fitting the sensorgrams with the two-state reaction model, which we also previously used to describe the binding mechanism of gH 7.
The differential rate equations for this reaction model are reported, where RU 1 and RU 2 are the response units for the first and second steps, respectively; C A is the peptide concentration; RU max is the maximum peptide binding capacity or equilibrium binding response ; and k a1 , k d1 , k a2 , and k d2 are the association and dissociation rate constants for the first and second steps, respectively.
CD spectra were recorded in a 1. Spectra were recorded and corrected for the background contributions by substracting blank samples. GUVs were used upon one day from preparation. The emerging fluorescence was detected by the Orca flash 2.
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The key for their ability to function as vectors is the membrane distortion caused by their partitioning at the membrane interface; thereby, membranotropic peptides modify the biophysical properties of phospholipid membranes; nonetheless, the exact mechanisms of insertion into and modification of target cell membranes are still unknown 1 , 2 , 3 , 4. Rice has always been stored up by several civilizations over the years as offerings for the gods and nourishment for human beings. Scientific Reports menu. Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process. Overall, the important role that NBA-containing structures play in biomedicine, as well as the possibility to realize new DNA- and RNA-binding nucleopeptides, prompted us to realize and investigate a new nucleobase-decorated artificial peptide, whose repeating unit is composed of a dipeptide moiety, based on the L -diaminopropionic acid L -Dap , connected via a short linker to the thymine nucleobase Figure 1. Historical Collection.
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