Background: An unknown mechanism promotes trans interactions by the GRASP homotypic membrane tethers rather than unproductive cis interactions. Results: Neutron reflection shows that the myristoylated GRASP domain has a fixed, upright orientation on the membrane incompatible with cis interactions. Conclusion: Myristoylation restricts the orientation of the protein on the membrane to favor interactions in trans. Significance: Orientation of membrane proteins is functionally significant and may be regulated by myristoylation.

Pore formation by membrane-active antimicrobial peptides is a classic strategy of pathogen inactivation through disruption of membrane biochemical gradients. It remains unknown why some membrane-active peptides also inhibit enveloped viruses, which do not depend on biochemical gradients. Here, we employ a label-free biosensing approach based on simultaneous quartz crystal microbalance-dissipation and ellipsometry measurements in order to investigate how a pore-forming, virucidal peptide destabilizes lipid vesicles in a surface-based experimental configuration. A key advantage of the approach is that it enables direct kinetic measurement of the surface-bound peptide-to-lipid (P:L) ratio. Comprehensive experiments involving different bulk peptide concentrations and biologically relevant membrane compositions support a unified model that membrane lysis occurs at or above a critical P:L ratio, which is at least several-fold greater than the value corresponding to the onset of pore formation. That is consistent with peptide-induced pores causing additional membrane strain that leads to lysis of highly curved membranes. Collectively, the work presents a new model that describes how peptide-induced pores may destabilize lipid membranes through a membrane strain-related lytic process, and this knowledge has important implications for the design and application of membrane-active peptides.

Due to their small size, nanoparticles (NPs) have the ability to penetrate cell membranes, and are therefore classified as potential human carcinogens. NP insertion into targeted cells also proves beneficial for drug delivery and gene therapy applications, prompting a need to more thoroughly characterize NP/ membrane interactions. Polystyrene NPs with modifications in size, surface functionalization and detergent conditions were introduced to a Langmuir lipid monolayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), a model of the outer leaflet of the cell membrane. At bilayer equivalent pressure, aminated and carboxylated NPs showed appreciable monolayer insertion whereas plain NPs solubilized the phospholipid layer, removing it from the air/water interface. Detergent added to a NP solution typically prevents particle aggregation, but the amphiphilic character of surfactant may also affect cell membrane interactions. Detergent-free NP solutions interact differently compared to those containing detergent, resulting in monolayer destruction. To examine the role of charged detergents, sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (DTAB) surfactants were introduced. Solutions composed of surfactant and NP functionalized with groups of the same charge showed appreciable interactions with the monolayer. Solutions of NPs and surfactants with opposing functional group charge aggregated, preventing authentic interactions. NP solutions with a higher concentration (1 wt%) of detergent initially showed increased insertion into the monolayer, suggesting cooperative behavior between NP and surfactant. The behavior of these NPs with the monolayer has two distinct regimes -an initial insertion event followed by monolayer destruction which is suggestive of interplay between kinetic and thermodynamic control.

Mammalian Golgi Reassembly and Stacking Proteins (GRASP) self-interact to organize the Golgi apparatus into an elongated membrane network. Localized at the surfaces of apposed membranes by simultaneous binding of a Golgiassociated protein and insertion of an N-terminal myristate, GRASP proteins tether bilayers through trans complexes. We investigated the mechanism that drives trans pairing, given that unproductive cis interactions within a single membrane are otherwise favored. We established an assay that quantifies GRASP-dependent membrane tethering in vitro and used neutron reflection to determine the structure of the GRASP/membrane complex. In the assay, we substituted a C-terminal His-tag interaction with Ni 2þ -NTA-functionalized lipid for the normal protein-mediated membrane contact. Hence, fluorescent, NTA-labeled liposomes binds to NTA-doped sparsely-tethered lipid bilayer membranes (stBLMs [1]) only if the added GRASP construct formed trans complexes between the two. While Ni 2þ chelation was sufficient to bind non-myristoylated GRASP to membranes, myristoylation increased GRASP tethering efficiency significantly. Neutron reflection showed that nonmyristoylated GRASP exhibited no preferential orientation on stBLMs. In contrast, the tethering-competent myristoylated GRASP exhibited a specific orientation, deduced using the GRASP crystal structure [2]. From these neutron results, a model for surface-ligated GRASP could be derived, and docking studies suggest a structure of the membrane-tethering GRASP complex. These results indicate that myristoylation restricts the orientation of GRASP to favor trans complexes for membrane tethering.