Secondary structure as a determinant of the rheological properties of peptide monolayers: a comparative study between the membrane disrupting peptides Melittin (-helix) and A1-40/2 (-sheet).

CARUSO, B, AMBROGGIO EE, N. WILKE, FIDELIO GD

CORDOBA

Jornada; Nano-Córdoba 2014, Jornadas sobre nanociencias y nanotecnología; 2014

The rheological study of interfaces covered with proteins have been receiving interest from many origins with their characterization including determination of the viscous and elastic components of the shear and dilatational responses. Among them, the shear behavior is of particular interest because it is associated with a 2D network that can arise as a consequence of intermolecular interactions (disulfide bridges, repulsive interactions at close packing or hydrogen bonding) or interdomain interactions. However, in spite of the wide amount of reports describing the shear behavior of proteins, the relative contributions of each of the different types of interactions has not been clearly depicted yet. In the present work, we choose to study spread monolayers of the membrane disrupting peptides, Melittin (Mel) and Ab1-40/42 whose structural properties allowed us to analyze the effect of the secondary structure on the shear properties of peptides monolayers. These peptides present well defined secondary structure when spread at the air-water interface. PM-IRRAS studies of spread Mel monolayers at pH 5.6-6 have shown that if forms preferentially alfa-helix, whereas Ab1-40 has been shown to develop monolayers with a prevalence of beta -sheet structure when spread in the air-water interface. Furthermore, Mel was also studied at pH 11, because previous studies have shown that its surface stability against lateral compression is increased at alkaline conditions, which may be interpreted as a consequence of an increase in beta-sheet content. Two rheological techniques were used, providing different advantages: 1) oscillatory and anisotropic compression of the monolayer allow two separate both elastic and viscous components of the dilatational and shear moduli and 2) although microrheological (diffusion of micron-sized beads embedded in the monolayer) determinations cannot separate viscous and elastic contributions this technique provides more sensibility. Both Ab1-40 and Ab1-42 peptides exhibit interfacial rheological properties characterized by a marked shear response. These monolayers presented a high microrheological shear response even at low surface pressures and exhibit high shear elasticity indicative of a solid network. Furthermore, this correlated with a theta-solvent condition, indicating poorer peptide solvation at the interface compared to Mel. In contrast, Mel monolayers exhibit a different rheological behaviour: no shear moduli was detected using oscillatory technique. The higher sensibility of the microrheological technique allowed to observe that bead diffusion was dependent on pH. At a determined lateral pressure, the microrheological shear response of Mel monolayers were higher at alkaline pH. This is correlated with the known high lateral stability of Mel at alkaline pH. FT-IR measurements indicate a change in the secondary structure of Mel with an increase in bulk pH, towards beta-sheet structure. Although Mel at high pH become more stable, it didn´t exhibit a shear elasticity comparable to Ab1-40/2. However, the diffusion of particles in this monolayer was intermediate between the alfa-helix Mel and the beta-sheet Ab1-40/2. Taken together, a) the that fact Mel monolayers exceeded the surface concentration predicted for the onset of shear as a consequence of steric jamming and b) the occurrence of a shear modulus in the b-sheet Ab1-40/2, even though its monolayers are less densely packed than the alfa-helix Mel, suggest that interactions other than steric jamming are responsible for such behaviour in these peptides. A network of electrostatic interactions cannot explain the observed differences since Ab1-40/2 are neutral whereas Mel have a +5 net charge at pH5.6 and +1 at pH11. Clearly, the interactions most evident for these systems are the hydrogen bonds manifesting in both secondary structures: beta-sheet being able to form an infinite planar network.