Molecular Transport and Growth of Lipid Vesicles Exposed to Antimicrobial Peptides

It is well-known that lipids constituting the cytoplasmic membrane undergo continuous reorganization to maintain the appropriate composition important for the integrity of the cell. The transport of lipids is controlled by mainly membrane proteins, but also spontaneous lipid transport between leaflets, lipid “flip–flop”, occurs. These processes do not only occur spontaneously under equilibrium, but also promote structural rearrangements, morphological transitions, and growth processes. It has previously been shown that intravesicular lipid “flip–flop” and intervesicular lipid exchange under equilibrium can be deduced indirectly from contrast variation time-resolved small-angle neutron scattering (TR-SANS) where the molecules are “tagged” using hydrogen/deuterium (H/D) substitution. In this work, we show that this technique can be extended to simultaneously detect changes in the growth and the lipid “flip–flop” and exchange rates induced by a peptide additive on lipid vesicles consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), d-DMPC (1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and small amounts of DMPE-PEG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy­(polyethylene glycol)-2000]). Changes in the overall size were independently monitored using dynamic light scattering (DLS). We find that the antimicrobial peptide, indolicidin, accelerates lipid transport and additionally induces limited vesicular growth. Moreover, in TR-SANS experiments using partially labeled lipid mixtures to separately study the kinetics of the lipid components, we show that, whereas peptide addition affects both lipids similarly, DMPG exhibits faster kinetics. We find that vesicular growth is mainly associated with peptide-mediated lipid reorganization that only slightly affects the overall exchange kinetics. This is confirmed by a TR-SANS experiment of vesicles preincubated with peptide showing that after pre-equilibration the kinetics are only slightly slower

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

Micelle Stabilization via Entropic Repulsion: Balance of Force Directionality and Geometric Packing of Subunit

Nanoparticles, 10–30 nm in size, have shown great prospects as nanocarriers for drug delivery. We designed amphiphiles based on 3-helix peptide-PEG conjugate forming 15 nm micelles (defined as “3-helix micelles”) with good in vivo stability. Here, we investigated the effect of the site of PEG conjugation on the kinetic stability and showed that the conjugation site affects the PEG chain conformation and the overall molecular architecture of the subunit. Compared to the original design where the PEG chain is located in the middle of the 3-helix bundle, micelle kinetic stability was reduced when the PEG chain was attached near the N-terminus (<i>t</i>_1/2 = 35 h) but was enhanced when the PEG chain was attached near the C-terminus (<i>t</i>_1/2 = 80 h). Quantitative structural and kinetic analysis suggest that the kinetic stability was largely dictated by the combined effects of entropic repulsion associated with PEG chain conformation and the geometric packing of the trimeric subunits. The modular design approach coupled with a variety of well-defined protein stucture and functional polymers will significantly expand the utility of these materials as nanocarriers to meet current demands in nanomedine

A Small-Angle X‑ray Scattering Study of α‑helical Bundle-Forming Peptide–Polymer Conjugates in Solution: Chain Conformations

As a new family of soft materials, peptide/protein–polymer conjugates can lead to a wide range of potential biological and nonbiological applications. The performance of these materials depends on the protein structure and phase behavior arising from a balance between the enthalpic interactions of the components and surrounding media as well as the entropic contribution associated with polymer chain deformation. There is a great need to perform structural studies in solution that systematically investigate the polymer chain conformation upon linkage to a peptide or protein so as to evaluate how polymers affect the protein structure of the biomolecule and, consequently, its functionality. Combinations of a range of factors including low contrast, weak scattering signals in dilute solutions as well as difficulties in separating the component scattering contributions, pose significant challenges to structural characterization. Here we present a synchrotron small-angle X-ray scattering (SAXS) study of two model helix bundle forming peptide–polymer conjugates and show that with analytical modeling of the scattering intensity detailed structural information on both peptide structure and polymer conformation can be extracted. The peptide–poly­(ethylene glycol) (PEG) conjugates are based on peptides that self-associate to form well-defined 3- or 4-helix bundles and the PEG chain is covalently linked either to the end or the side of the peptide (i.e. end- or side-conjugation). Using a simplified analytical geometrical body form factor model, where the peptide–polymer bundles are modeled as parallel cylinders with attached Gaussian chains, a quantitative description of the scattering behavior can be reached. On the basis of the simplified structural model, the protein tertiary structures, i.e., the α-helix bundle, remains largely intact and maintains its oligomeric state but exhibits slight swelling in solution with respect to the crystal structure. The PEG chain conformation appears to slightly depend on the conjugate architecture. In terms of the chain dimension represented by <i>R</i>_<i>g</i>, the end-conjugated PEG exhibit similar value as compared to free PEG for the molecular weight studied (2 kDa). For the side-conjugates our simple scattering model seems to indicate a systematically slightly lower values for <i>R</i>_<i>g</i>, i.e., a slight compression, in particular for the highest molecular weight (5 kDa). However, considering the limitations of the model and experimental uncertainties, further investigations, such as neutron scattering, is needed to illustrate detailed chain conformation. The present studies can be extended to other peptide–polymer or protein–polymer hybrid systems to extract information on both protein structure and polymer chain conformation. This work will thus provide valuable guidance to understand their structure and phase behavior using X-ray and neutron scattering

Solution Structural Characterization of Coiled-Coil Peptide–Polymer Side-Conjugates

Detailed structural characterization of protein–polymer conjugates and understanding of the interactions between covalently attached polymers and biomolecules will build a foundation to design and synthesize hybrid biomaterials. Conjugates based on simple protein structures are ideal model system to achieve these ends. Here we present a systematic structural study of coiled-coil peptide–poly­(ethylene glycol) (PEG) side-conjugates in solution, using circular dichroism, dynamic light scattering, and small-angle X-ray scattering, to determine the conformation of conjugated PEG chains. The overall size and shape of side-conjugates were determined using a cylindrical form factor model. Detailed structural information of the covalently attached PEG chains was extracted using a newly developed model where each peptide–PEG conjugate was modeled as a Gaussian chain attached to a cylinder, which was further arranged in a bundle-like configuration of three or four cylinders. The peptide–polymer side-conjugates were found to retain helix bundle structure, with the polymers slightly compressed in comparison with the conformation of free polymers in solution. Such detailed structural characterization of the peptide–polymer conjugates, which elucidates the conformation of conjugated PEG around the peptide and assesses the effect of PEG on peptide structure, will contribute to the rational design of this new family of soft materials

Understanding Peptide Oligomeric State in Langmuir Monolayers of Amphiphilic 3‑Helix Bundle-Forming Peptide-PEG Conjugates

Coiled-coil peptide–polymer conjugates are an emerging class of biomaterials. Fundamental understanding of the coiled-coil oligomeric state and assembly process of these hybrid building blocks is necessary to exert control over their assembly into well-defined structures. Here, we studied the effect of peptide structure and PEGylation on the self-assembly process and oligomeric state of a Langmuir monolayer of amphiphilic coiled-coil peptide–polymer conjugates using X-ray reflectivity (XR) and grazing-incidence X-ray diffraction (GIXD). Our results show that the oligomeric state of PEGylated amphiphiles based on 3-helix bundle-forming peptide is surface pressure dependent, a mixture of dimers and trimers was formed at intermediate surface pressure but transitions into trimers completely upon increasing surface pressure. Moreover, the interhelical distance within the coiled-coil bundle of 3-helix peptide-PEG conjugate amphiphiles was not perturbed under high surface pressure. Present studies provide valuable insights into the self-assembly process of hybrid peptide–polymer conjugates and guidance to develop biomaterials with controlled multivalency of ligand presentation

Self-Concentration and Interfacial Fluctuation Effects on the Local Segmental Dynamics of Nanostructured Diblock Copolymer Melts

Self-Concentration and Interfacial Fluctuation Effects on the Local Segmental Dynamics of Nanostructured Diblock Copolymer Melt

Internal Structure of 15 nm 3‑Helix Micelle Revealed by Small-Angle Neutron Scattering and Coarse-Grained MD Simulation

3-Helix micelles (3HM) formed by self-assembly of peptide–polymer conjugate amphiphiles have shown promise as a nanocarrier platform due to their long-circulation, deep tumor penetration, selective accumulation in tumor, and ability to cross the blood-brain barrier (BBB) for glioblastoma therapy. There is a need to understand the structural contribution to the high in vivo stability and performance of 3HM. Using selective deuteration, the contrast variation technique in small-angle neutron scattering, and coarse-grained molecular dynamics simulation, we determined the spatial distribution of each component within 3HM. Our results show a slightly deformed polyethylene glycol (PEG) conformation within the micelle that is radially offset from its conjugation site toward the exterior of the micelle and a highly solvated shell. Surprisingly, ∼85 v/v % of 3HM is water, unusually higher than any micellar nanocarrier based on our knowledge. The result will provide important structural insights for future studies to uncover the molecular origin of 3HM’s in vivo performance, and development of the nanocarriers

Kinetic Pathway of the Cylinder-to-Sphere Transition in Block Copolymer Micelles Observed in Situ by Time-Resolved Neutron and Synchrotron Scattering

Here we present an in situ study of the nonequilibrium cylinder-to-sphere morphological transition kinetics on the millisecond range in a model block copolymer micelle system revealing the underlying mechanism and pathways of the process. By employing the stopped-flow mixing technique, the system was rapidly brought (≈100 μs) deep into the instability region, and the kinetics was followed on the time scale of milliseconds using both time-resolved small-angle neutron and X-ray scattering (TR-SANS and TR-SAXS, respectively). Due to the difference in contrast and resolution, SAXS and SANS provide unique complementary information. Our analysis shows that the morphological transition is characterized by a single rate constant indicating a two-state model where the transition proceeds through direct decomposition (fragmentation) of the cylinders without any transient intermediate structures. The cylindrical segments formed in the disintegration process subsequently grow into spherical micelles possibly through the molecular exchange mechanism until near equilibrium micelles are formed. The observation of a two-step kinetic mechanism, fluctuation-induced fragmentation and ″ripening″ processes, provides unique insight into the nonequilibrium behavior of block copolymer micelles in dilute solutions

Transformation from Globular to Cylindrical Mixed Micelles through Molecular Exchange that Induces Micelle Fusion

Transformations between different micellar morphologies in solution induced by changes in composition, salt, or temperature are well-known phenomena; however, the understanding of the associated kinetic pathways is still limited. Especially for mixed surfactant systems, the micelles can take a very wide range of structures, depending on the surfactant packing parameter and other thermodynamic conditions. Synchrotron-based small-angle X-ray scattering (SAXS) in combination with fast mixing using a stopped-flow apparatus can give direct access to the structural kinetics on a millisecond time scale. Here, this approach is used to study the formation of cylindrical micelles after mixing two solutions with globular micelles of the nonionic surfactant dodecyl maltoside (DDM) and the anionic surfactant sodium dodecyl sulfate (SDS), respectively. Two separate processes were identified: (i) a transition in micellar shell structure, interpreted as exchange of surfactant molecules resulting in mixed globular micelles, and subsequently, (ii) fusion into larger, cylindrical structures