Abstract
Polybia-MP1 (IDWKKLLDAAKQIL-NH2), a helical peptide extracted from the venom of a Brazilian wasp, has broad-spectrum antimicrobial activities without being hemolytic or cytotoxic. This peptide has also displayed anticancer activity against cancer cell cultures. Despite its high selectivity, MP1 has an unusual low net charge (Q = +2). The aspartic residue (D2) in the N-terminal region plays an important role in its affinity and selectivity; its substitution by asparagine (D2N mutant) led to a less selective peptide. Aiming to explore the importance of this residue for the peptides’ affinity, we compared the zwitterionic and anionic vesicle adsorption activity of Polybia-MP1 versus its D2N mutant and also mastoparan X (MPX). The adsorption, electrostatic, and conformational free energies were assessed by circular dichroism (CD) and fluorescence titrations using large unilamellar vesicles (LUVs) at the same conditions in association with measurement of the zeta potential of LUVs in the presence of the peptides. The adsorption free energies of the peptides, determined from the partition coefficients, indicated higher affinity of MP1 to anionic vesicles compared with the D2N mutant and MPX. The electrostatic and conformational free energies of MP1 in anionic vesicles are less favorable than those found for the D2N mutant and MPX. Therefore, the highest affinity of MP1 to anionic vesicles is likely due to other energetic contributions. The presence of D2 in MP1 makes these energetic components 1.2 and 1.5 kcal/mol more favorable compared with the D2N mutant and MPX, respectively.
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Acknowledgments
J.R.N. acknowledges the financial support from São Paulo Research Foundation (FAPESP, grant # 2011/11640-5). J.R.N. and M.S.P. are researchers of CNPq. N.B.L. has a PhD grant from CAPES, and D.S.A. has a fellowship from São Paulo Research Foundation (FAPESP, grant # 2012/08147-8).
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249_2014_945_MOESM1_ESM.jpg
Fig. S1 a Circular dichroism spectra of D2N mutant at 10 μM. Spectra recorded in the absence and in the presence of 0.5 mM of PC/PG (70:30) and PC LUVs in 5 mM Tris-H3BO3, 0.15 M NaF, 0.5 mM Na2EDTA, pH 7.5, at 25 °C. b Hydrodynamic diameters (D H) obtained from the diffusion coefficient (D) using the Stokes–Einstein equation, D H = kT/3πηD, where k is the Boltzmann constant and T is the absolute temperature by dynamic light scattering of the peptide titration. c Molar ellipticity normalized at 222 nm as a function of lipid concentration. D2N-MP1 at 10 µM titrated with zwitterionic (open symbols) and anionic (closed symbols) lipids. Solid lines: nonlinear fit using Eq. 2-b (JPEG 186 kb)
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Fig. S2 a Circular dichroism spectra of MPX at 10 μM. Spectra recorded in the absence and in the presence of 0.5 mM of PC/PG (70:30) and PC LUVs in 5 mM Tris-H3BO3, 0.15 M NaF, 0.5 mM Na2EDTA, pH 7.5, at 25 °C. b Hydrodynamic diameters (D H) obtained from the diffusion coefficient (D) using the Stokes–Einstein equation, D H = kT/3πηD, where k is the Boltzmann constant and T is the absolute temperature by dynamic light scattering of the peptide titration. c Molar ellipticity normalized at 222 nm as a function of lipid concentration. MPX at 10 µM titrated with zwitterionic (open symbols) and anionic (closed symbols) lipids. Solid lines: nonlinear fit using Eq. 2-b (JPEG 189 kb)
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Fig. S3 a Fluorescence emission spectra of D2N mutant at 5 μM in 5 mM Tris-H3BO3, 0.15 M NaF, 0.5 mM Na2EDTA, pH 7.5, at 25 °C in the presence of PC LUVs, from bottom to top at 325 nm: 0, 0.2, 0.29, 0.38, 0.475, 0.65, 0.9, and 1.3 mM of lipid. b Normalized fluorescence emission intensity as a function of lipid concentration. D2N-MP1 at 5 µM titrated with zwitterionic (open symbols) and anionic (closed symbols) lipids. Solid lines: nonlinear fit using Eq. 4 (JPEG 830 kb)
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Fig. S4 a Fluorescence emission spectra of MPX at 5 μM in 5 mM Tris-H3BO3, 0.15 M NaF, 0.5 mM Na2EDTA, pH 7.5, at 25 °C in the presence of PC LUVs, from bottom to top at 325 nm: 0, 0.2, 0.29, 0.38, 0.475, 0.65, 0.9, and 1.3 mM of lipid. b Normalized fluorescence emission intensity as a function of lipid concentration. MPX at 5 µM titrated with zwitterionic (open symbols) and anionic (closed symbols) lipids. Solid lines: nonlinear fit using Eq. 4 (JPEG 813 kb)
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Leite, N.B., dos Santos Alvares, D., de Souza, B.M. et al. Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles. Eur Biophys J 43, 121–130 (2014). https://doi.org/10.1007/s00249-014-0945-1
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DOI: https://doi.org/10.1007/s00249-014-0945-1