Membrane proteins represent a significant frontier in structural biology they are ubiquitous in nature and perform a variety of tasks that help govern cellular activity. Their structure, insertion mechanisms, and function largely depend on the interactions between peptide-lipid domains and the hydrating water. Therefore, the dynamics of the membrane-associated water and its interaction with embedded proteins remain some of the most fundamental issues in biological physics today. Single-supported lipid bilayers (SSLBs) provide model systems for investigating their structural and dynamical properties via atomic force microscopy (AFM) and quasielastic neutron scattering (QENS), respectively. QENS measurements on SSLBs comprised of zwitterionic (DMPC) and anionic (DMPG) lipids reveal vastly different freezing/melting behavior of their hydration water, while also elucidating various types of membrane-associated water characterized by their translational diffusion rates. Moreover, results from temperature-dependent neutron diffraction measurements on SSLBs have established a correlation between the formation of various crystalline ice structures and freezing/melting transitions observed in the elastic component of their QENS spectra, thereby confirming the various growth modes of the membrane-associated ice. We have since enhanced the complexity and biological relevance of such systems by incorporating the antimicrobial peptide, melittin, into a DMPC membrane. On monitoring the incoherent elastic neutron intensity as a function of temperature from melittin-treated DMPC membranes, we observe an abrupt freezing transition of the associated water not seen in the bare membrane case. Moreover, the change in elastic intensity of this freezing step increases in proportion to peptide concentration, suggesting that water could be freezing onto membrane-bound melittin. In addition to bulk-like water present in the sample, analysis of the quasielastic spectra collected provides evidence of a second water type that diffuses more slowly and freezes at a higher temperature than the bulk-like water. Furthermore, in situ AFM studies reveal the formation of dimple-like features on the surfaces of such membranes when melittin concentrations exceed 0.5 [mu]M. These changes induced in the bilayer have been interpreted as aggregates of membrane-bound melittin responsible for the altered freezing behavior and dynamics of the hydration water. An unexpected time dependence of the elastically-scattered neutron intensity was observed when membranes of DMPC treated with 0.5 [mu]M melittin were annealed in the temperature range 325 K less than T less than 340 K, an effect not present in DMPC membranes treated with other melittin concentrations. These results are consistent with the slowing down of hydrogen nuclei and anchoring of surface-bound melittin peptides while interacting with DMPC membranes.
Hydration Water Freezing in Single Supported Lipid Bilayers
