Heiko Heerklotz

Triton Promotes Domain Formation in Lipid Raft Mixtures

Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds.

Titration calorimetry of surfactant-membrane partitioning and membrane solubilization.

The interaction of surfactants with membranes has been difficult to monitor since most detergents are small organic molecules without spectroscopic markers. The development of high sensitivity isothermal titration calorimetry (ITC) has changed this situation distinctly. The insertion of a detergent into the bilayer membrane is generally accompanied by a consumption or release of heat which can be measured fast and reliably with modern titration calorimeters. It is possible to determine the full set of thermodynamic parameters, i.e., the partitioning enthalpy, the partitioning isotherm, the partition coefficient, the free energy, and the entropy of transfer. The application of ITC to the following problems is described: (i) measurement of the critical micellar concentration (CMC) of pure detergent solutions; (ii) analysis of surfactant-membrane partitioning equilibria, including asymmetric insertion; and (iii) membrane-surfactant phase diagrams. Finally, the thermodynamic parameters derived for non-ionic detergents are discussed and the affinity for micelle formation is compared with membrane incorporation.

Interactions of surfactants with lipid membranes

Surfactants are surface-active, amphiphilic compounds that are water-soluble in the micro- to millimolar range, and self-assemble to form micelles or other aggregates above a critical concentration. This definition comprises synthetic detergents as well as amphiphilic peptides and lipopeptides, bile salts and many other compounds. This paper reviews the biophysics of the interactions of surfactants with membranes of insoluble, naturally occurring lipids. It discusses structural, thermodynamic and kinetic aspects of membrane-water partitioning, changes in membrane properties induced by surfactants, membrane solubilisation to micelles and other phases formed by lipid-surfactant systems. Each section defines and derives key parameters, mentions experimental methods for their measurement and compiles and discusses published data. Additionally, a brief overview is given of surfactant-like effects in biological systems, technical applications of surfactants that involve membrane interactions, and surfactant-based protocols to study biological membranes.

The sensitivity of lipid domains to small perturbations demonstrated by the effect of Triton.

The hypothesis of lipid rafts describes functional domains in biological membranes. It is often assumed that rafts form by spontaneous de-mixing of certain lipids and that they can be isolated as detergent-resistant membrane particles (DRMs) using the detergent Triton X-100 (TX). Here, we present a model that describes the process of domain formation in membranes in the presence and in the absence of TX. We measure the interactions between TX and an equimolar mixture of sphingomyelin (SM), cholesterol (Cho), and 1-palmitoyl-2-oleoyl-3-sn-glycero-phosphatidylcholine (POPC) (1:1:1, mol) by means of isothermal titration calorimetry. Comparison with pure POPC membranes reveals a very unfavorable interaction between TX and SM/Cho, which causes a substantial tendency to segregate these molecules into separate, DRM-like (SM-rich) and fluid (TX-rich), domains. If rafts are indeed formed by spontaneous de-mixing of PC and SM/Cho, they must be very sensitive, and perturbations caused by techniques used to study rafts could lead to misleading results. If, however, rafts are much more stable than PC-SM-Cho domains, there must be an unknown raft stabilizer. Subtle changes of such a promoter could serve to modulate raft function.

Detergent-like action of the antibiotic peptide surfactin on lipid membranes

Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.

Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization

Diguanylate cyclases (DGCs) are key enzymes of second messenger signaling in bacteria. Their activity is responsible for the condensation of two GTP molecules into the signaling compound cyclic di-GMP. Despite their importance and abundance in bacteria, catalytic and regulatory mechanisms of this class of enzymes are poorly understood. In particular, it is not clear if oligomerization is required for catalysis and if it represents a level for activity control. To address this question we perform in vitro and in vivo analysis of the Caulobacter crescentus diguanylate cyclase PleD. PleD is a member of the response regulator family with two N-terminal receiver domains and a C-terminal diguanylate cyclase output domain. PleD is activated by phosphorylation but the structural changes inflicted upon activation of PleD are unknown. We show that PleD can be specifically activated by beryllium fluoride in vitro, resulting in dimerization and c-di-GMP synthesis. Cross-linking and fractionation experiments demonstrated that the DGC activity of PleD is contained entirely within the dimer fraction, confirming that the dimer represents the enzymatically active state of PleD. In contrast to the catalytic activity, allosteric feedback regulation of PleD is not affected by the activation status of the protein, indicating that activation by dimerization and product inhibition represent independent layers of DGC control. Finally, we present evidence that dimerization also serves to sequester activated PleD to the differentiating Caulobacter cell pole, implicating protein oligomerization in spatial control and providing a molecular explanation for the coupling of PleD activation and subcellular localization.

Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin

Titin forms an intrasarcomeric filament system in vertebrate striated muscle, which has elastic and signaling properties and is thereby central to mechanotransduction. Near its C‐terminus and directly preceding a kinase domain, titin contains a conserved pattern of Ig and FnIII modules (IgA168‐IgA169‐FnIIIA170, hereby A168‐A170) that recruits the E3 ubiquitin‐ligase MuRF‐1 to the filament. This interaction is thought to regulate myofibril turnover and the trophic state of muscle. We have elucidated the crystal structure of A168‐A170, characterized MuRF‐1 variants by circular dichroism (CD) and SEC‐MALS, and studied the interaction of both components by isothermal calorimetry, SPOTS blots, and pull‐down assays. This has led to the identification of the molecular determinants of the binding. A168‐A170 shows an extended, rigid architecture, which is characterized by a shallow surface groove that spans its full length and a distinct loop protrusion in its middle point. In MuRF‐1, a C‐terminal helical domain is sufficient to bind A168‐A170 with high affinity. This helical region predictably docks into the surface groove of A168‐A170. Furthermore, pull‐down assays demonstrate that the loop protrusion in A168‐A170 is a key mediator of MuRF‐1 recognition. Our findings indicate that this region of titin could serve as a target to attempt therapeutic inhibition of MuRF‐1‐mediated muscle turnover, where binding of small molecules to its distinctive structural features could block MuRF‐1 access.—Mrosek, M., Labeit, D., Witt, S., Heerklotz, H., von Castelmur, E., Labeit, S., Mayans, O. Molecular determinants for the recruitment of the ubiquitin‐ligase MuRF‐1 onto M‐line titin FASEB J. 21, 1383–1392 (2007)

The Enthalpy of Acyl Chain Packing and the Apparent Water-Accessible Apolar Surface Area of Phospholipids

The energetics of phospholipid aggregation depend on the apparent water-accessible apolar surface area (ASAap), ordering effects of the chains, and headgroup interactions. We quantify the enthalpy and entropy of these interactions separately. For that purpose, the thermodynamics of micelle formation of lysophosphatidylcholines (LPCs, chains C10, C12, C14, and C16) and diacylphosphatidylcholines (DAPCs, chains C5, C6) and C7) are studied using isothermal titration calorimetry. The critical micelle concentration (CMC) values are 90, 15, and 1.9 mM (C5-C7-DAPC) and 6.8, 0.71, 0.045, and 0.005 mM (LPCs). The group contributions per methylene of DeltaDeltaG(0) = -3.1 kJ/mol and DeltaDeltaC(P) = -57 J/(mol. K) for LPCs agree with literature data on hydrocarbons and amphiphiles. An apparent deviation of DAPCs (-2.5 kJ/mol, 45 J/(mol. K)) is due to an intramolecular interaction between the two chains, burying 20% of the surface. The chain/chain interaction enthalpies in a micelle core are by approximately -2 kJ/(mol) per methylene group more favorable than in bulk hydrocarbons. We conclude that the impact of the chain conformation and packing on the interaction enthalpy is very pronounced. It serves to explain a variety of effects reported on membrane binding. Interactions within the water-accessible region show considerable DeltaH, but almost no DeltaG(0). The heat capacity changes suggest about three methylene groups (ASAap approximately 100 A2) per LPC remain exposed to water in a micelle (DAPC: 2 CH2/70 A2).

Classifying surfactants with respect to their effect on lipid membrane order.

We propose classifying surfactants with respect to their effect on membrane order, which is derived from the time-resolved fluorescence anisotropy of DPH. This may help in understanding why certain surfactants, including biosurfactants such as antimicrobial lipopeptides and saponins, often show a superior performance to permeabilize and lyse membranes and/or a better suitability for membrane protein solubilization. Micelle-forming surfactants induce curvature stress in membranes that causes disordering and, finally, lysis. Typical detergents such as C(12)EO(8), octyl glucoside, SDS, and lauryl maltoside initiate membrane lysis after reaching a substantial, apparently critical extent of disordering. In contrast, the fungicidal lipopeptides surfactin, fengycin, and iturin from Bacillus subtilis QST713 as well as digitonin, CHAPS, and lysophosphatidylcholine solubilize membranes without substantial, overall disordering. We hypothesize they disrupt the membrane locally due to a spontaneous segregation from the lipid and/or packing defects and refer to them as heterogeneously perturbing. This may account for enhanced activity, selectivity, and mutual synergism of antimicrobial biosurfactants and reduced destabilization of membrane proteins by CHAPS or digitonin. Triton shows the pattern of a segregating surfactant in the presence of cholesterol.

The microcalorimetry of lipid membranes

Insight into the forces governing a system is essential for understanding its behaviour and function. Calorimetric investigations provide a wealth of information that is not, or is hardly, available by other methods. This paper reviews calorimetric approaches and assays for the study of lipid vesicles (liposomes) and biological membranes. With respect to the instrumentation, differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), isothermal titration calorimetry (ITC) and water sorption calorimetry are considered. Applications of these techniques to lipid systems include the measurement of thermodynamic parameters and a detailed characterization of the thermotropic, barotropic, and lyotropic phase behaviour. The membrane binding or partitioning of solutes (proteins, peptides, drugs, surfactants, ions, etc) can also be quantified. Many calorimetric assays are available for studying the effect of proteins and other additives on membranes, characterizing non-ideal mixing, domain formation, stability, curvature strain, permeability, solubilization, and fusion. Studies of membrane proteins in lipid environments elucidate lipid–protein interactions in membranes. The systems are described in terms of enthalpic and entropic forces, equilibrium constants, heat capacities, partial volume changes etc, shedding light also on the stability of structures and the molecular origin and mechanism of structural changes.