markedly with increasing temperature (Figure 4, middle traces in panels C and E). Other properties attesting to a remarkable conformational stability exhibited by the ShB peptide in the presence of anionic phospholipid vesicles are that the absorbance at 1623 cm-1 i) recovers quite efficiently upon cooling of previously heat-denatured samples (Figure 4, upper traces in panels C and E); ii) is fairly insensitive to increasing the ionic strength of the aqueous media, unless the vesicles are preincubated, prior to the addition of the peptide, with higher than 1M NaCl which efficiently prevents the conformational event; iii) is insensitive to the presence of low concentrations of non-anionic detergents such as octyl-glucoside, unless the vesicles are completely disrupted by the detergents at concentrations above their critical micellar concentration and iv) is insensitive to the presence of millimolar concentrations of EDTA (data not shown).
    As to the mutant ShB-L7E peptide (Figure 4, panels D and F), the 1623 cm-1 * structure component can be observed only in PG vesicles (panel F), at a moderately high peptide concentration and only in the virtual absence of EDTA (below *20 *M) and even then, it is much less heat-stable than that seen in the ShB peptide under similar condition


Leyenda FIGURE2:

Changes in the gel to liquid crystal phase transition enthalpies of DMPA (panel A, circles) or DMPG (panel B, triangles) large multilamellar vesicles in the presence of increasing concentrations of either ShB (closed symbols) or ShB-L7E (open symbols) peptides, as determined by differential scanning calorimetry of the indicated phospholipid/peptide mixtures in 10 mM Hepes, pH 7.0, 100 mM NaCl. Error bars are within the size of the symbols used to plot the experimental results. In the absence of added peptides, the estimated phase transition enthalpies in vesicles made from pure DMPA or DMPG were 5.4 and 6.5 Kcal/mol of phospholipid, respectively. Panel C is a plot of the observed *H/*H0 from above (transition enthalpies of either DMPA (closed circles) or DMPG (closed triangles) determined in the presence (*H) and in the absence(*H0) of added ShB peptide) versus the ShB peptide/phospholipid ratios. In spite of the heterogeneity arising from the use of data from DMPA and DMPG samples altogether, a linear extrapolation of such plot indicates that the addition of ShB peptide in this samples at a peptide/phospholipid ratio of approximately 0.3 will completely avoid the occurrence of the cooperative lipid phase transition.

Leyenda FIGURE3:


    pH dependence of the observed gel to liquid crystal phase transition enthalpy of large multilamellar DMPG vesicles, as determined by differential scanning calorimetry. Symbols correspond to DMPG vesicles alone (open hexagons) and in the presence of either ShB (closed triangles) or ShB-L7E (open triangles) peptides, both at a peptide/DMPG molar ratio of 0.1. The buffers used at each of the indicated pH's were: pH 5.0, 25 mM citrate, 50 mM phosphate, 20 mM NaCl, 130 mM KCl; pH 6.0, 10 mM Mes, 20 mM NaCl, 130 mM KCl; pH 7.0, 10 mM phosphate, 20 mM NaCl, 130 mM KCl; pH 7.5, 10 mM phosphate, 20 mM NaCl, 130 mM KCl; pH 8.0, 10 mM Epps, 20 mM NaCl, 130 mM KCl; pH 8.5, 10 mM Epps, 20 mM NaCl, 130 mM KCl.