Helix Fraying May Stabilize Transmembrane Alpha Helices

Abstract

membrane and the atomic structure has been resolved by X-ray crystallography. The hydrophobic thickness of the protein observed in the structure 29.7A is considerably thinner than that predicted for the membrane 35.5A suggesting a hydrophobic mismatch. This hydrophobic mismatch is expected to aid protein-protein interactions and result in lipid sorting. Furthermore the surface of the protein has several cationic groups that could result in specific interactions with anionic lipid species. In order to investigate the local environment of Aquaporin Z in native E. coli membranes, and so understand the degree of lipid sorting and local protein-protein interactions, we have isolated Styrene-Maleic Acid Lipid Particles. These particles are believed to contain small portions of membrane isolated by the detergent action of the polymer. We have analysed particles isolated from E.coli membranes and containing Aquaporing Z for size, lipid and protein content. We have compared these experimental results to results obtained from coarse grained molecular dynamic simulations. These results are interpreted in terms of the interactions between the aquaporin and its environment. Synaptotagmins are membrane trafficking proteins that contain two C-terminal C2 domains, C2A and C2B. In eight of the seventeen mammalian isoforms, the C2 domains serve as calcium-dependent membrane binding motifs that trigger exocytosis upon cellular calcium influx. C2 domains from the different isoforms display different kinetics and affinities for membranes, which reflect their functions in a variety of different cell types. For example, synaptotagmin-7 (Syt7) is mainly active in slow secretion events such as insulin release. Previous work in our lab has shown that the C2A domain of human Syt7 has membrane association and dissociation rates that are~2 and~60-fold slower than the corresponding domain of Syt1, an isoform involved in rapid neurotransmitter release [Brandt DS, et al., Biochemistry; 51(39):7654-7664.] The purpose of this study is to investigate the balance of forces governing the stronger membrane binding of Syt7 C2A. We have used kinetic measurements of association and dissociation to assess membrane affinity, while systematically varying buffer composition, liposome size, lipid composition, and mutant forms of the protein domain. The results indicate that this domain's unusually strong membrane binding strength is due to a combination of electrostatics and the hydrophobic effect. In particular, dissociation rates are governed mainly by the hydrophobic effect, as seen by slower dissociation in the presence of the kosmotropic agent trehalose. Furthermore, the F167 residue in the Syt7 C2A domain acts as a secondary hydrophobic anchor for the membranedocked protein domain, as its mutation to methionine (as in Syt1) results in a reproducible 2-fold increase in off rate.