PMC

Percent calcein leakage from LUV in buffer at 15 min after addition of X31 (•) and ccX31 (○) at pH 7.0 (a), and X31 (•) and ccX31 (○) at pH 5.0 (b), as a function of peptide-to-lipid ratio. The LUVs were composed of SOPC/SOPS (99:1 mol %).

CD spectra of ccX31 in pH 5.0 and pH 7.0 buffers. The peptide was added from a concentrated stock solution (∼4.1 mM in 50% aqueous methanol) to the buffers to give a final peptide concentration of 53 μM.

Percent peptide-induced lipid mixing induced at 15 min after addition of X31 (•) and ccX31 (○) at pH 7.0 (a), and X31 (•) and ccX31 (○) at pH 5.0 (b), as a function of peptide-to-lipid ratio. The unlabeled LUVs and doubly labeled LUVs were composed of SOPC/SOPS (99:1 mol %) and SOPC/SOPS/NBD-PE/LRh-PE (97.8:1:0.6:0.6 mol %), respectively.

(A) Schematic of a transfer pipette experiment. Experiments are conducted in glass chambers on the stage of an inverted microscope. (B) Photographs of an SOPC vesicle (i) before exposure to peptide, (ii) increasing projection length due to peptide adsorption, and (iii) lysing of vesicle due to peptide.

(Bottom panel) Sedimentation equilibrium analysis of ccX31 in 10 mM MES, 10 mM MOPS at pH 7.0 with 1% (v/v) dimethyl sulfoxide. The loading concentration of ccX31 was ∼30 μM. Data collected at 40,000 rpm is shown fitted with trimer species. The top panel shows the residual of the fit.

Titration of ccX31 and X31 peptides with SOPC/NBD-PE LUV (98.6:1.4 mol %) as monitored by fluorescence. Fluorescence emissions were measured for LUV at 400 μM in buffer with increasing amounts of added peptide, and the intensities in the range of 500–560 nm were integrated. Intensity ratios (F/F₀) are plotted where F₀ is the integrated fluorescence intensity measured in the absence of peptide. •, X31 at pH 7; ▪, X31 at pH 5; ○, ccX31 at pH 7; □, ccX31 at pH 5.

Experimental kinetics of calcein release induced by ccX31 and X31. Various amounts of ccX31 and X31 were added to vesicles containing ∼40 mM encapsulated calcein in the presence of X31 at pH 7.0 (a), X31 at pH 5.0 (b), ccX31 at pH 7.0 (c), and ccX31 at pH 5.0 (d). The LUVs were composed of SOPC/SOPS (99:1 mol %). The kinetics of the leakage were followed by monitoring the increase of the fluorescence intensity at 520 nm and are plotted versus time.

Experimental kinetics of lipid mixing induced by ccX31 and X31. Various amounts of ccX31 and X31 were added to vesicles containing ∼40 mM encapsulated calcein in the presence of X31 at pH 7.0 (a), X31 at pH 5.0 (b), ccX31 at pH 7.0 (c), and ccX31 at pH 5.0 (d). The kinetics of the leakage were followed by monitoring the increase of the fluorescence intensity at 533 nm and are plotted versus time. The unlabeled LUVs and doubly labeled LUVs were composed of SOPC/SOPS (99:1 mol %) and SOPC/SOPS/NBD-PE/LRh-PE (97.8:1:0.6:0.6 mol %), respectively.

Representative aspiration data plots, showing (a) change in projection length (L) versus time and (b) change in area expansion (α) versus time for 1 μM ccX31 at pH 5. Note that there is no permeation phase before lysis, which is representative of all experiments with ccX31 at pH 5. If there was a permeation phase after the initial adsorption phase, as seen with the X31 peptide (), then once a pore forms the projection length would begin to decrease as the vesicle swells until it falls. Lysis of the vesicle without the additional strain introduced by swelling indicates that ccX31 induces a dramatic drop in the tension strength of the membrane. The lipid vesicles are SOPC/SOPS (99.5:0.5 mol %).

Representative projection length (L) versus time data for 10 μM X31 at pH 5 (data courtesy of M. Longo). Note the characteristic shape of the curve, compared to that of ccX31 (). First there is an increase in L as peptide adsorbs and inserts into the membrane, slightly increasing the apparent surface area of the vesicle. Then, when one or more pores form, there is a transition to the permeation phase. The vesicle swells as glucose and water flow into the vesicle and the larger entrapped sucrose cannot flow out. The projection length decreases as the vesicle swells until failure. This permeation phase is not seen in the ccX31 data. In the presence of ccX31, vesicles lyse without the additional strain introduced by swelling, indicating that ccX31 induces a dramatic drop in the tensile strength of the membrane.