Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

Purified myelin lipids display a critical mixing point at low surface pressure.

Lipids extracted from Purified Myelin Membranes (LPMM) were spread as monomolecular films at the air/aqueous interface. The films were visualized by Brewster Angle Microscopy (BAM) at different lateral pressures (π) and ionic environments. Coexistence of Liquid-Expanded (LE) and cholesterol-enriched (CE) rounded domains persisted up to π ≈ 5 mN/m but the monolayers became homogeneous at higher surface pressures. Before mixing, the domains distorted to non-rounded domains. We experimentally measured the line tension (λ) for the lipid monolayers at the domain borders by a shape relaxation technique using non-homogeneous electric fields. Regardless of the subphase conditions, the obtained line tensions are of the order of pN and tended to decrease as lateral pressure increased toward the mixing point. From the mean square displacement of nested trapped domains, we also calculated the dipole density difference between phases (µ). A non-linear drop was detected in this parameter as the mixing point is approached. Here we quantitively evaluated the π-dependance of both parameters with proper power laws in the vicinity of the critical mixing surface pressure, and the exponents showed to be consistent with a critical phenomenon in the two-dimensional Ising universality class. This idea of bidimensionality was found to be compatible only for simplified lipidic systems, while for whole myelin monolayers, that means including proteins, no critical mixing point was detected. Finally, the line tension values were related with the thickness differences between phases (Δt) near the critical point.

Phase Diagram of Purified CNS Myelin Reveals Continuous Transformation between Expanded and Compacted Lamellar States.

Purified myelin membranes (PMMs) are the starting material for biochemical studies, from individual components up to the isolation of detergent-resistant membrane (DRM) fractions or detergent-insoluble glycosphingolipid (DIG) fractions, which are commonly believed to resemble physiological lipid rafts. The normal DIG isolation protocol involves the extraction of lipids under moderate cooling. The isolation of PMMs also involves the cooling of myelin as well as exposure to low ionic strength (IS). Here, we addressed the combined influence of cooling and IS on the structure of PMMs. The phase behaviour was investigated by small angle X-ray diffraction. Analysis of the diffraction peaks revealed the lamellar periodicity ( d ), the number of periodically correlated bilayers ( N ), and the relatives fractions of each phase. Departure from physiological conditions induced a phase separation in myelin. The effect of monovalent and divalent ions was also compared at equivalent IS, showing a differential effect, and phase diagrams for both ion types were established-Ca2+ induced the well-known over-compacted phase, but additionally we also found an expanded phase at low IS. Na+ promoted phase separation, and also induced over-compaction at sufficiently high IS. Finally, exploring the whole phase diagram, we found evidence for the direct isothermal transformation from the expanded to the compacted phase, suggesting that both phases could in fact originate from the identical primary lateral phase separation, whereas the apparent difference lies in the inter-bilayer interaction that is modulated by the ionic milieu.

Correction: Cooling induces phase separation in membranes derived from isolated CNS myelin.

[This corrects the article DOI: 10.1371/journal.pone.0184881.].

Cooling induces phase separation in membranes derived from isolated CNS myelin.

Purified myelin membranes (PMMs) are the starting material for biochemical analyses such as the isolation of detergent-insoluble glycosphingolipid-rich domains (DIGs), which are believed to be representatives of functional lipid rafts. The normal DIGs isolation protocol involves the extraction of lipids under moderate cooling. Here, we thus address the influence of cooling on the structure of PMMs and its sub-fractions. Thermodynamic and structural aspects of periodic, multilamellar PMMs are examined between 4°C and 45°C and in various biologically relevant aqueous solutions. The phase behavior is investigated by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Complementary neutron diffraction (ND) experiments with solid-supported myelin multilayers confirm that the phase behavior is unaffected by planar confinement. SAXS and ND consistently show that multilamellar PMMs in pure water become heterogeneous when cooled by more than 10-15°C below physiological temperature, as during the DIGs isolation procedure. The heterogeneous state of PMMs is stabilized in physiological solution, where phase coexistence persists up to near the physiological temperature. This result supports the general view that membranes under physiological conditions are close to critical points for phase separation. In presence of elevated Ca2+ concentrations (> 10 mM), phase coexistence is found even far above physiological temperatures. The relative fractions of the two phases, and thus presumably also their compositions, are found to vary with temperature. Depending on the conditions, an "expanded" phase with larger lamellar period or a "compacted" phase with smaller lamellar period coexists with the native phase. Both expanded and compacted periods are also observed in DIGs under the respective conditions. The observed subtle temperature-dependence of the phase behavior of PMMs suggests that the composition of DIGs is sensitive to the details of the isolation protocol.

Refractive index and thickness determination in Langmuir monolayers of myelin lipids.

Langmuir monolayers at the air/water interface are widely used as biomembrane models and for amphiphilic molecules studies in general. Under controlled intermolecular organization (lateral molecular area), surface pressure, surface potential, reflectivity (R) and other magnitudes can be precisely determined on these planar monomolecular films. However, some physical parameters such as the refractive index of the monolayer (n) still remain elusive. The refractive index is very relevant because (in combination with R) it allows for the determination of the thickness of the film. The uncertainties of n determine important errors that propagate non-linearly into the calculation of monolayers thickness. Here we present an analytical method for the determination of n in monolayers based on refractive index matching. By using a Brewster angle microscopy (BAM) setup and monolayers spread over subphases with variable refractive index (n2), a minimum in R is search as a function of n2. In these conditions, n equals n2. The results shown correspond to monolayers of myelin lipids. The n values remain constant at 1.46 upon compression and equals the obtained value for myelin lipid bilayers in suspension. The values for n and R allow for the determination of thickness. We establish comparisons between these thicknesses for the monolayer and those obtained from two X-ray scattering techniques: 1) GIXOS for monolayers at the air/water interface and 2) SAXS for bilayers in bulk suspension. This allows us to conclude that the thickness that we measure by BAM includes the apolar and polar headgroup regions of the monolayer.

CNS myelin structural modification induced in vitro by phospholipases A2.

Myelin is the self-stacked membrane surrounding axons; it is also the target of several pathological and/or neurodegenerative processes like multiple sclerosis. These processes involve, among others, the hydrolytic attack by phospholipases. In this work we describe the changes in isolated myelin structure after treatment with several secreted PLA2 (sPLA2), by using small angle x-ray scattering (SAXS) measurements. It was observed that myelin treated with all the tested sPLA2s (from cobra and bee venoms and from pig pancreas) preserved the lamellar structure but displayed an enlarged separation between membranes in certain zones. Additionally, the peak due to membrane asymmetry was clearly enhanced. The coherence length was also lower than the non-treated myelin, indicating increased disorder. These SAXS results were complemented by Langmuir film experiments to follow myelin monolayer hydrolysis at the air/water interface by a decrease in electric surface potential at different surface pressures. All enzymes produced hydrolysis with no major qualitative difference between the isoforms tested.