Pathways of Membrane Solubilization: A Structural Study of Model Lipid Vesicles Exposed to Classical Detergents.

Understanding the pathways of solubilization of lipid membranes is of high importance for their use in biotechnology and industrial applications. Although lipid vesicle solubilization by classical detergents has been widely investigated, there are few systematic structural and kinetic studies where different detergents are compared under varying conditions. This study used small-angle X-ray scattering to determine the structures of lipid/detergent aggregates at different ratios and temperatures and studied the solubilization in time using the stopped-flow technique. Membranes composed of either of two zwitterionic lipids, DMPC or DPPC, and their interactions with three different detergents, sodium dodecyl sulfate (SDS), n-dodecyl-beta-maltoside (DDM), and Triton X-100 (TX-100), were tested. The detergent TX-100 can cause the formation of collapsed vesicles with a rippled bilayer structure that is highly resistant to TX-100 insertion at low temperatures, while at higher temperatures, it partitions and leads to the restructuring of vesicles. DDM also causes this restructuring into multilamellar structures at subsolubilizing concentrations. In contrast, partitioning of SDS does not alter the vesicle structure below the saturation limit. Solubilization is more efficient in the gel phase for TX-100 but only if the cohesive energy of the bilayer does not prevent sufficient partitioning of the detergent. DDM and SDS show less temperature dependence compared to TX-100. Kinetic measurements reveal that solubilization of DPPC largely occurs through a slow extraction of lipids, whereas DMPC solubilization is dominated by fast and burst-like solubilization of the vesicles. The final structures obtained seem to preferentially be discoidal micelles where the detergent can distribute in excess along the rim of the disc, although we do observe the formation of worm- and rodlike micelles in the case of solubilization of DDM. Our results are in line with the suggested theory that bilayer rigidity is the main factor influencing which aggregate is formed.

Understanding the Structural Pathways for Lipid Nanodisc Formation: How Styrene Maleic Acid Copolymers Induce Membrane Fracture and Disc Formation.

Lipid nanodiscs formed by mixtures of styrene maleic acid (SMA) copolymers and lipid membranes are important tools for studying membrane proteins in many biotechnological applications. However, molecular interactions leading up to their formation are not well understood. Here, we elucidate the nanodisc formation pathways for SMA/lipid vesicle mixtures using small-angle X-ray scattering (SAXS) that allows detailed in situ nanostructural information. SMA copolymer that is initially aggregated in solution inserts its styrene units into the lipid bilayer hydrocarbon region, leading to fractures in the membrane. The initial copolymer-lipid interactions observed in the vesicles are also present in the formed discs, with excess copolymer distributed along the normal of the bilayer. The size and SMA distribution in the resulting discs strongly depend on the temperature, lipid/copolymer ratio, and lipid type. We find that the solubilization limit increases for membranes above the melting point, suggesting that defects in gel-like lipid membranes play a significant role in membrane fracturing and nanodisc formation. These findings provide unique insights into the formation of nanodiscs as well as into the microscopic mechanism of solubilization, which plays an important role in many applications and products ranging from household goods to biotechnology and medicine.

High-Resolution Crystal Structures Elucidate the Molecular Basis of Cholera Blood Group Dependence.

Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1-1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera.

Resolving the structural interactions between antimicrobial peptides and lipid membranes using small-angle scattering methods: the case of indolicidin.

Using small angle X-ray and neutron scattering (SAXS/SANS) and detailed theoretical modelling we have elucidated the structure of the antimicrobial peptide, indolicidin, and the interaction with model lipid membranes of different anionic lipid compositions mimicking typical charge densities found in the cytoplasmic membrane of bacteria. First, we show that indolicidin displays a predominantly disordered, random chain conformation in solution with a small fraction (≈1%) of fiber-like nanostructures that are not dissolved at higher temperatures. The peptide is shown to strongly interact with the membranes at all charge densities without significantly perturbing the lipid bilayer structure. Instead, the results show that indolicidin inserts into the outer leaflet of the lipid vesicles causing a reduced local order of the lipid packing. This result is supported by an observed change in the melting point of the lipids upon addition of the peptide, as seen by differential scanning calorimetry experiments. The peptide does not to our observation affect the thickness of the membrane or form distinct structural pores in the membrane at physiologically relevant concentrations as has been previously suggested as an important mode of action. Finally, using sophisticated contrast variation SANS, we show that the peptide does not affect the random lateral distribution of anionic lipids in the membrane. Together, these results demonstrate that the structural aspects of the mode of action of antimicrobial peptides can be elucidated in detail using SAS techniques with liposomes as model systems.

Micelle kinetics of photoswitchable surfactants: Self-assembly pathways and relaxation mechanisms.

HYPOTHESIS: A key question in the kinetics of surfactant self-assembly is whether exchange of unimers or fusion/fission of entire micelles is the dominant pathway. In this study, an isomerizable surfactant is used to explore fundamental out-of-equilibrium kinetics and mechanisms for growth and dissolution of micelles. EXPERIMENTS: The kinetics of cationic surfactant 4-butyl-4'-(3-trimethylammoniumpropoxy)-phenylazobenzene was studied using molecular dynamics simulations. The fusion and exchange processes were investigated using umbrella sampling. Equilibrium states were validated by comparison with small-angle X-ray scattering data. The photo-isomerization event was simulated by modifying the torsion potential of the photo-responsive group to emulate the trans-to-cis transition. FINDINGS: Micelle growth is dominated by unimer exchange processes, whereas, depending on the conditions, dissolution can occur both through fission and unimer expulsion. Fusion barriers increase steeply with the aggregation number making this an unlikely pathway to equilibrium for micelles of sizes that fit with the experimental data. The barriers for unimer expulsion remain constant and are much lower for unimer insertion, making exchange more likely at high aggregation. When simulating photo-conversion events, both fission and a large degree of unimer expulsion can occur depending on the extent of the out-of-equilibrium stress that is put on the system.

Beyond the standard model of solubilization: Non-ionic surfactants induce collapse of lipid vesicles into rippled bilamellar nanodiscs.

HYPOTHESIS: Although solubilization of lipid membranes has been studied extensively, questions remain regarding the structural pathways and metastable structures involved. This study investigated whether the non-ionic detergent Triton X-100 follows the classical solubilization pathway or if intermediate nanostructures are formed. EXPERIMENTS: Small angle X-ray and neutron scattering (SAXS/SANS) was used in combination with transmission electron cryo-microscopy and cryo-tomography to deduce the structure of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles and Triton X-100. Time-resolved SAXS and dynamic light scattering were used to investigate the kinetics of the process. FINDINGS: Upon addition of moderate detergent amounts at low temperatures, the lipid vesicles implode into ordered rippled bilamellar disc structures. The bilayers arrange in a ripple phase to accommodate packing constraints caused by inserted TX-100 molecules. The collapse is suggested to occur through a combination of water structure destabilization by detergents flipping across the membrane and osmotic pressure causing interbilayer attraction internally. The subsequently induced ripples then stabilize the aggregates and prevent solubilization, supported by the observation that negatively charged vesicles undergo a different pathway upon TX-100 addition, forming large bicelles. The findings demonstrate the richness in assembly pathways of simple lipids and detergents and stimulate considerations for the use of certain detergents in membrane solubilization.

Beyond structural models for the mode of action: How natural antimicrobial peptides affect lipid transport.

HYPOTHESIS: Most textbook models for antimicrobial peptides (AMP) mode of action are focused on structural effects and pore formation in lipid membranes, while these deformations have been shown to require high concentrations of peptide bound to the membrane. Even insertion of low amounts of peptides in the membrane is hypothesized to affect the transmembrane transport of lipids, which may play a key role in the peptide effect on membranes. EXPERIMENTS: Here we combine state-of-the-art small angle X-ray/neutron scattering (SAXS/SANS) techniques to systematically study the effect of a broad selection of natural AMPs on lipid membranes. Our approach enables us to relate the structural interactions, effects on lipid exchange processes, and thermodynamic parameters, directly in the same model system. FINDINGS: The studied peptides, indolicidin, aurein 1.2, magainin II, cecropin A and LL-37 all cause a general acceleration of essential lipid transport processes, without necessarily altering the overall structure of the lipid membranes or creating organized pore-like structures. We observe rapid scrambling of the lipid composition associated with enhanced lipid transport which may trigger lethal signaling processes and enhance ion transport. The reported membrane effects provide a plausible canonical mechanism of AMP-membrane interaction and can reconcile many of the previously observed effects of AMPs on bacterial membranes.