The conformation of human phospholipid scramblase 1, as studied by infrared spectroscopy. Effects of calcium and detergent.

Human phospholipid scramblase 1 (SCR) is a membrane protein that catalyzes the transmembrane (flip-flop) motion of phospholipids. It can also exist in a non membrane-bound form in the nucleus, where it modulates several aspects of gene expression. Catalysis of phospholipid flip-flop requires the presence of millimolar Ca2+, and occurs in the absence of ATP. Membrane-bound SCR contains a C-terminal α-helical domain embedded in the membrane bilayer. The latter domain can be removed giving rise to a stable truncated mutant SCRΔ that is devoid of scramblase activity. In order to improve our understanding of SCR structure infrared spectra have been recorded of both the native and truncated forms, and the effects of adding Ca2+, or removing detergent, or thermally denaturing the protein have been observed. Under all conditions the main structural component of SCR/SCRΔ is a ß-sheet. Removing the C-terminal 28 aa residues, which anchor SCR to the membrane, leads to a change in tertiary structure and an increased structural flexibility. The main effect of Ca2+ is an increase in the α/ß ratio of secondary structure components, with a concomitant increase in the proportion of non-periodic structures. At least in SCRΔ, detergent (Zwittergent 3-12) decreases the structural flexibility, an effect somewhat opposite to that of increasing temperature. Thermal denaturation is affected by Ca2+, detergent, and by the presence or absence of the C-terminal domain, each of them influencing in different ways the denaturation pattern.

Model systems of precursor cellular membranes: long-chain alcohols stabilize spontaneously formed oleic acid vesicles.

Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid.

Interdomain Ca(2+) effects in Escherichia coli alpha-haemolysin: Ca(2+) binding to the C-terminal domain stabilizes both C- and N-terminal domains.

alpha-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca(2+) concentrations. Previous studies have shown that Ca(2+) binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca(2+) using native HlyA, a truncated form of HlyADeltaN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of alpha-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca(2+) binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca(2+)-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca(2+) in HlyA may well be common to other members of this family.

Sphingosine-1-phosphate as an amphipathic metabolite: its properties in aqueous and membrane environments.

Sphingosine-1-phosphate (S1P) is currently considered to be an important signaling molecule in cell metabolism. We studied a number of relevant biophysical properties of S1P, using mainly Langmuir balance, differential scanning calorimetry, (31)P-NMR, and infrared (IR) spectroscopy. We found that, at variance with other, structurally related sphingolipids that are very hydrophobic, S1P may occur in either an aqueous dispersion or a bilayer environment. S1P behaves in aqueous media as a soluble amphiphile, with a critical micelle concentration of approximately 12 muM. Micelles give rise to larger aggregates (in the micrometer size range) at and above a 1 mM concentration. The aggregates display a thermotropic transition at approximately 60 degrees C, presumably due to the formation of smaller structures at the higher temperatures. S1P can also be studied in mixtures with phospholipids. Studies with dielaidoylphosphatidylethanolamine (DEPE) or deuterated dipalmitoylphosphatidylcholine (DPPC) show that S1P modifies the gel-fluid transition of the glycerophospholipids, shifting it to lower temperatures and decreasing the transition enthalpy. Low (<10 mol %) concentrations of S1P also have a clear effect on the lamellar-to-inverted hexagonal transition of DEPE, i.e., they increase the transition temperature and stabilize the lamellar versus the inverted hexagonal phase. IR spectroscopy of natural S1P mixed with deuterated DPPC allows the independent observation of transitions in each molecule, and demonstrates the existence of molecular interactions between S1P and the phospholipid at the polar headgroup level that lead to increased hydration of the carbonyl group. The combination of calorimetric, IR, and NMR data allowed the construction of a temperature-composition diagram ("partial phase diagram") to facilitate a comparative study of the properties of S1P and other related lipids (ceramide and sphingosine) in membranes. In conclusion, two important differences between S1P and ceramide are that S1P stabilizes the lipid bilayer structure, and physiologically relevant concentrations of S1P can exist dispersed in the cytosol.

Triton X-100 partitioning into sphingomyelin bilayers at subsolubilizing detergent concentrations: effect of lipid phase and a comparison with dipalmitoylphosphatidylcholine.

We examined the partitioning of the nonionic detergent Triton X-100 at subsolubilizing concentrations into bilayers of either egg sphingomyelin (SM), palmitoyl SM, or dipalmitoylphosphatidylcholine. SM is known to require less detergent than phosphatidylcholine to achieve the same extent of solubilization, and for all three phospholipids solubilization is temperature dependent. In addition, the three lipids exhibit a gel-fluid phase transition in the 38-41 degrees C temperature range. Experiments have been performed at Triton X-100 concentrations well below the critical micellar concentration, so that only detergent monomers have to be considered. Lipid/detergent mol ratios were never <10:1, thus ensuring that the solubilization stage was never reached. Isothermal titration calorimetry, DSC, and infrared, fluorescence, and (31)P-NMR spectroscopies were applied in the 5-55 degrees C temperature range. The results show that, irrespective of the chemical nature of the lipid, DeltaG degrees of partitioning remained in the range of -27 kJ/mol lipid in the gel phase and of -30 kJ/mol lipid in the fluid phase. This small difference cannot account for the observed phase-dependent differences in solubilization. Such virtually constant DeltaG degrees occurred as a result of the compensation of enthalpic and entropic components, which varied with both temperature and lipid composition. Consequently, the observed different susceptibilities to solubilization cannot be attributed to differential binding but to further events in the solubilization process, e.g., bilayer saturability by detergent or propensity to form lipid-detergent mixed micelles. The data here shed light on the relatively unexplored early stages of membrane solubilization and open new ways to understand the phenomenon of membrane resistance toward detergent solubilization.

The calcium-binding C-terminal domain of Escherichia coli alpha-hemolysin is a major determinant in the surface-active properties of the protein.

alpha-Hemolysin (HlyA) from Escherichia coli is a protein toxin (1024 amino acids) that targets eukaryotic cell membranes, causing loss of the permeability barrier. HlyA consists of two main regions, an N-terminal domain rich in amphipathic helices, and a C-terminal Ca(2+)-binding domain containing a Gly- and Asp-rich nonapeptide repeated in tandem 11-17 times. The latter is called the RTX domain and gives its name to the RTX protein family. It had been commonly assumed that membrane interaction occurred mainly if not exclusively through the amphipathic helix domain. However, we have cloned and expressed the C-terminal region of HlyA, containing the RTX domain plus a few stabilizing sequences, and found that it is a potent surface-active molecule. The isolated domain binds Ca(2+) with about the same affinity (apparent K(0.5) approximately 150 microM) as the parent protein HlyA, and Ca(2+) binding induces in turn a more compact folding with an increased proportion of beta-sheet structure. Both with and without Ca(2+) the C-terminal region of HlyA can interact with lipid monolayers spread at an air-water interface. However, the C-terminal domain by itself is devoid of membrane lytic properties. The present results can be interpreted in the light of our previous studies that involved in receptor binding a peptide in the C-terminal region of HlyA. We had also shown experimentally the distinction between reversible membrane adsorption and irreversible lytic insertion of the toxin. In this context, the present data allow us to propose that both major domains of HlyA are directly involved in membrane-toxin interaction, the nonapeptide repeat, calcium-binding RTX domain being responsible for the early stages of HlyA docking to the target membrane.