The mechanism of the interaction between the cell-penetrating peptide transportan 10 (tp10) and phospholipid membranes was investigated. Tp10 induces graded release of the contents of phospholipid vesicles. The kinetics of peptide association with vesicles and peptide-induced dye efflux from the vesicle lumen were examined experimentally by stopped-flow fluorescence. The experimental kinetics were analyzed by directly fitting to the data the numerical solution of mathematical kinetic models. A very good global fit was obtained using a model in which tp10 binds to the membrane surface and perturbs it because of the mass imbalance thus created across the bilayer. The perturbed bilayer state allows peptide monomers to insert transiently into its hydrophobic core and cross the membrane, until the peptide mass imbalance is dissipated. In that transient state tp10 "catalyzes" dye efflux from the vesicle lumen. These conclusions are consistent with recent reports that used molecular dynamics simulations to study the interactions between peptide antimicrobials and phospholipid bilayers. A thermodynamic analysis of tp10 binding and insertion in the bilayer using water-membrane transfer hydrophobicity scales is entirely consistent with the model proposed. A small bilayer perturbation is both necessary and sufficient to achieve very good agreement with the model, indicating that the role of the lipids must be included to understand the mechanism of cell-penetrating and antimicrobial peptides.

Nucleic acid binding proteins are generally viewed as either specific or non-specific, depending on characteristics of their binding sites in DNA or RNA 1,2 . Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure, or both [1][2][3][4] . Proteins that bind to sites devoid of defined sequence or structure signatures are considered non-specific 1,2,5 . Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly non-specific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures 6 . Here, we systematically examine substrate binding by the apparently non-specific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the tRNA processing ribonucleoprotein enzyme RNase P from E. coli. The protein binds 5′ leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HiTS-Kin) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants strikingly resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The kinetics of carboxyfluorescein efflux induced by the amphipathic peptide delta-lysin from vesicles of porcine brain sphingomyelin (BSM), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol (Chol) were investigated as a function of temperature and composition. Sphingomyelin (SM)/Chol mixtures form a liquid-ordered (L(o)) phase whereas POPC exists in the liquid-disordered (L(d)) phase at ambient temperature. delta-Lysin binds strongly to L(d) and poorly to L(o) phase. In BSM/Chol/POPC vesicles the rate of carboxyfluorescein efflux induced by delta-lysin increases as the POPC content decreases. This is explained by the increase of delta-lysin concentration in L(d) domains, which enhances membrane perturbation by the peptide. Phase separations in the micrometer scale have been observed by fluorescence microscopy in SM/Chol/POPC mixtures for some SM, though not for BSM. Thus, delta-lysin must detect heterogeneities (domains) in BSM/Chol/POPC on a much smaller scale. Advantage was taken of the inverse variation of the efflux rate with the L(d) content of BSM/Chol/POPC vesicles to estimate the L(d) fraction in those mixtures. These results were combined with differential scanning calorimetry to obtain the BSM/Chol/POPC phase diagram as a function of temperature.

We have been examining the mechanism and kinetics of the interactions of a selected set of peptides with phospholipid membranes, in a quantitative manner. This set was chosen to cover a broad range of physical-chemical properties and cell specificities. Mastoparan (masL) and mastoparan X (masX) are two similar peptides from the venoms of the wasps Vespula lewisii and Vespa xanthoptera, respectively, and were chosen to complete the set. The rate constants for masX association with, and dissociation from membranes are reported here for the first time. The kinetics of dye efflux induced by both mastoparans from phospholipid vesicles were also examined and quantitatively analyzed. We find that masL and masX follow the same graded kinetic model that we previously proposed for the cell-penetrating peptide transportan 10 (tp10), but with different parameters. This comparison is relevant because tp10 is derived from masL by addition of a mostly nonpolar segment of 7 residues at the N-terminus. Tp10 is more active than the mastoparans toward phosphatidylcholine vesicles, but the mastoparans are more sensitive to the effect of anionic lipids. Furthermore, the Gibbs free energies of binding and insertion of the peptides calculated using the Wimley-White transfer scales are in good agreement with the values derived from our experimental data, and are useful for understanding peptide behavior.During the past several years, we have been examining the mechanisms of a set of antimicrobial, cytolytic, and cell-penetrating peptides using a unified approach that includes a quantitative analysis of their function on phospholipid large unilamellar vesicles (LUVs) 1 . In this manner, all peptides can be directly compared, and the origin of the differences and similarities in their mechanisms better rationalized. This set of peptides includes δ-lysin (1, 2), cecropin A (3), magainin 2 (4), and the cell-penetrating peptide transportan 10 (tp10) (5, 6), all of which form amphipathic α-helices when bound to membrane surfaces. These peptides vary in length from 21-37 residues, which is enough to span the lipid bilayer if inserted perpendicularly to the membrane surface, and their net charges vary from 0 to +7 at pH 7.5. Thus, they cover a broad range of physical-chemical properties. Tp10 is a man-made, 21-residue, chimeric construct obtained by linking the 6 residues of the neuropeptide galanin, through an extra lysine residue, to the 14 residues of mastoparan from Vespula lewisii, which becomes the C-terminal segment of tp10 (7,8). Previously, we investigated the kinetics of tp10 binding to phospholipid membranes, the kinetics of tp10-induced dye efflux from vesicles, and its mechanism of dye release (5). Tp10 causes graded release of lipid vesicle contents, and its interaction with membranes is consistent with peptide translocation across the bilayer (5), which is essential for its function (8). † This work was supported by National Institutes of Health Grant GM072507. *To whom correspondence should be addressed: Tel: (910) 962-...

The kinetics and thermodynamics of binding of transportan 10 (tp10) and four of its variants to phospholipid vesicles, and the kinetics of peptide-induced dye efflux, were compared. Tp10 is a 21-residue, amphipathic, cationic, cell-penetrating peptide similar to helical antimicrobial peptides. The tp10 variants examined include amidated and free peptides, and replacements of tyrosine by tryptophan. Carboxy-terminal amidation or substitution of tryptophan for tyrosine enhance binding and activity. The Gibbs energies of peptide binding to membranes determined experimentally and calculated from the interfacial hydrophobicity scale are in good agreement. The Gibbs energy for insertion into the bilayer core was calculated using hydrophobicity scales of residue transfer from water to octanol and to the membrane/water interface. Peptide-induced efflux becomes faster as the Gibbs energies for binding and insertion of the tp10 variants decrease. If anionic lipids are included, binding and efflux rate increase, as expected because all tp10 variants are cationic and an electrostatic component is added. Whether the most important effect of peptide amidation is the change in charge or an enhancement of helical structure, however, still needs to be established. Nevertheless, it is clear that the changes in efflux rate reflect the differences in the thermodynamics of binding and insertion of the free and amidated peptide groups.