The lyophilized pellets were resolubilized in 4 mL 12.5% acetonitrile, 0.1% TFA in water. Purification was carried out by reversed-phase HPLC Ultimate 3000 (Dionex, Sunnyvale, CA), monitoring peptide elution at 230 nm. Approximately 20 mg of the crude peptides were chromatographed

using an Onyx Monolithic C18 column (10 × 100 mm, 13 nm & 2 μm pore size) with a linear gradient of 0.1% TFA in water (v/v) and 0.85% TFA in acetonitrile (v/v) at a flow rate of 5 mL/min over 25 min. The fractions of interest were spotted onto a stainless steel MALDI plate and observed by MALDI-TOF (Applied Biosystems/MDS SCIEX, Foster City, CA). Fractions containing greater than 80% purity http://www.selleckchem.com/products/carfilzomib-pr-171.html were pooled and lyophilized. The Y-27632 nmr synthetic peptides were used in the assays below. Chloroform solution of asolectin was evaporated under N2 flow, rendering homogeneous films on round bottom flasks that were further dried under vacuum for at least 3 h. Films were hydrated at room temperature with buffer (Tris/H3BO3 5 mM, 0.5 mM Na2EDTA, 150 mM NaF, pH 7.5) to reach a final lipid concentration of 10 mg/mL and vortex mixed. SUVs were obtained after 50 min sonication (or until clear) with a tip sonicator in an ice/water

bath, under N2 flow; titanium debris was removed by centrifugation. SUVs were then submitted to 6 extrusions, at room temperature, through a 100 nm polycarbonate membrane followed by 11 extrusions through two stacked 50 nm polycarbonate membranes, using an Avanti mini-extruder. SUVs were kept under refrigeration and used in the same day of preparation. CD spectra were obtained at 20 μM peptide concentration in different environments: bi-distilled water, 5 mM Tris/H3BO3 buffer, pH 7.5, 8 mM sodium dodecylsulfate (SDS) solution (above critical micelle concentration), 40% v/v trifluoroethanol

(TFE)/water mixture, and in the presence Rutecarpine of 100 and 250 μg/mL asolectin vesicles. TFE solutions are known inductors of helical structures and micellar SDS as well as vesicles are membrane mimetic environments with anionic character, a feature common to bacterial membranes (Yeaman and Yount, 2003). At 40% TFE or at micellar concentration of SDS solutions (8 mM) they tend to induce the maximum observable values (Prates et al., 2004). In the presence of asolectin vesicles saturation was found at 250 μg/mL concentration. CD spectra were recorded from 260 to 203 or 190 nm (depending on signal-to-noise ratio) with a Jasco-710 spectropolarimeter (JASCO International Co. Ltd., Tokyo, Japan) which was routinely calibrated at 290.5 nm using d-10-camphorsulfonic acid solution. Spectra were acquired at 25 °C using 0.5-cm path length cell, averaged over eight scans, at a scan speed of 20 nm/min, bandwidth of 1.0 nm, 0.5 s response, and 0.2 nm resolution.