The effects on dielaidoylphosphatidylethanolamine (DEPE) bilayers of ceramides containing different N-acyl chains have been studied by differential scanning calorimetry small angle x-ray diffraction and (31)P-NMR spectroscopy. N-palmitoyl (Cer16), N-hexanoyl (Cer6), and N-acetyl (Cer2) sphingosines have been used. Both the gel-fluid and the lamellar-inverted hexagonal transitions of DEPE have been examined in the presence of the various ceramides in the 0-25 mol % concentration range. Pure hydrated ceramides exhibit cooperative endothermic order-disorder transitions at 93 degrees C (Cer16), 60 degrees C (Cer6), and 54 degrees C (Cer2). In DEPE bilayers, Cer16 does not mix with the phospholipid in the gel phase, giving rise to high-melting ceramide-rich domains. Cer16 favors the lamellar-hexagonal transition of DEPE, decreasing the transition temperature. Cer2, on the other hand, is soluble in the gel phase of DEPE, decreasing the gel-fluid and increasing the lamellar-hexagonal transition temperatures, thus effectively stabilizing the lamellar fluid phase. In addition, Cer2 was peculiar in that no equilibrium could be reached for the Cer2-DEPE mixture above 60 degrees C, the lamellar-hexagonal transition shifting with time to temperatures beyond the instrumental range. The properties of Cer6 are intermediate between those of the other two, this ceramide decreasing both the gel-fluid and lamellar-hexagonal transition temperatures. Temperature-composition diagrams have been constructed for the mixtures of DEPE with each of the three ceramides. The different behavior of the long- and short-chain ceramides can be rationalized in terms of their different molecular geometries, Cer16 favoring negative curvature in the monolayers, thus inverted phases, and the opposite being true of the micelle-forming Cer2. These differences may be at the origin of the different physiological effects that are sometimes observed for the long- and short-chain ceramides.
The molecular basis for the existence of the so-called “detergent-resistant membranes” has been explored. With that aim, vesicles composed of phosphatidylcholine, sphingolipid, and cholesterol were treated with the nonionic detergent Triton X-100 either at 4 °C or at 37 °C and tested for solubilization using turbidity and centrifugation methods. Bilayer fluidity was systematically measured as fluorescence anisotropy of a diphenylhexatriene derivative of phosphatidylcholine. Putative sphingomyelin−cholesterol interactions were explored using IR spectroscopy. The combined experimental evidence clearly indicates that these lipid mixtures are solubilized more easily at 4 °C than at 37 °C, that an increased membrane fluidity does not correlate with an easier solubilization, and that sphingomyelin−cholesterol interactions are essential for insolubility. Sphingolipids by themselves do not hinder detergent solubilization, and some of them, e.g., gangliosides, actually increase bilayer solubility in the presence of detergents. At least with some lipid compositions, there is a range of detergent concentrations at which partial solubilization occurs concomitantly with major changes in bilayer architecture (lysis and reassembly). Moreover, a nonsolubilized residue of composition phosphatidylcholine/sphingomyelin/cholesterol of ca. 1:1:1 (mole ratio) is recovered by centrifugation after detergent treatment of vesicles with very different original lipid compositions. These observations do not preclude the presence of liquid-ordered domains in the cell membrane but support the idea that the detergent-resistant membranes obtained after detergent treatments may well be the result of bilayer partial solubilization and reassembly, instead of corresponding precisely to structures pre-existing in the cell membrane.
We have studied the conformation of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), free in solution and bound to the DNA, by Fourier-transform infrared spectroscopy. The peptide belongs to the COOH-terminal domain of histone H1 0 (residues 99 -121) and is adjacent to the central globular domain of the protein. In aqueous (D 2 O) solution the amide I is dominated by component bands at 1643 cm ؊1 and 1662 cm ؊1 , which have been assigned to random coil conformations and turns, respectively. In accordance with previous NMR results, the latter component has been interpreted as arising in turn-like conformations in rapid equilibrium with unfolded states. The peptide becomes fully structured either in 90% trifluoroethanol (TFE) solution or upon interaction with the DNA. In these conditions, the contributions of turn (1662 cm ؊1 ) and random coil components virtually disappear. In TFE, the spectrum is dominated by the ␣-helical component (1654 cm ؊1 ). The band at 1662 cm ؊1 shifts to 1670 cm ؊1 , and has been assigned to the COOH-terminal TPKK motif in a more stable turn conformation. A band at 1637 cm ؊1 , also present in TFE, has been assigned to 3 10 helical structure. The amide I band of the complexes with the DNA retains the components that were attributed to 3 10 helix and the TPKK turn. In the complexes with the DNA, the ␣-helical component observed in TFE splits into two components at 1657 cm ؊1 and 1647 cm ؊1 . Both components are inside the spectral region of ␣-helical structures. Our results support the presence of inducible helical and turn elements, both sharing the character of DNA-binding motifs.
Interactions of palmitoylsphingomyelin with cholesterol in multilamellar vesicles have been studied over a wide range of compositions and temperatures in excess water by using electron spin resonance (ESR) spectroscopy. Spin labels bearing the nitroxide free radical group on the 5 or 14 C-atom in either the sn-2 stearoyl chain of phosphatidylcholine (predominantly 1-palmitoyl) or the N-stearoyl chain of sphingomyelin were used to determine the mobility and ordering of the lipids in the different phases. Two-component ESR spectra of the 14-position spin labels demonstrate the coexistence first of gel (L(beta)) and liquid-ordered (L(o)) phases and then of liquid-ordered and liquid-disordered (L(alpha)) phases, with progressively increasing temperature. These phase coexistences are detected over a limited range of cholesterol contents. ESR spectra of the 5-position spin labels register an abrupt increase in ordering at the L(alpha)-L(o) transition and a biphasic response at the L(beta)-L(o) transition. Differences in outer splitting between the C14-labeled sphingomyelin and phosphatidylcholine probes are attributed to partial interdigitation of the sphingomyelin N-acyl chains across the bilayer plane in the L(o) state. In the region where the two fluid phases, L(alpha) and L(o), coexist, the rate at which lipids exchange between phases (<<7 x 10(7) s(-)(1)) is much slower than translational rates in the L(alpha) phase, which facilitates resolution of two-component spectra.
It is important to establish the structural properties of linker histones to understand the role they play in chromatin higher order structure and gene regulation. Here, we use CD, NMR, and IR spectroscopy to study the conformation of the amino-terminal domain of histone H1°, free in solution and bound to the DNA. The NH 2 -terminal domain has little structure in aqueous solution, but it acquires a substantial amount of ␣-helical structure in the presence of trifluoroethanol (TFE). As in other H1 subtypes, the basic residues of the NH 2 -terminal domain of histone H1°are clustered in its COOH-terminal half. According to the NMR results, the helical region comprises the basic cluster (Lys 11 -Lys 20 ) and extends until Asp 23 . The fractional helicity of this region in 90% TFE is about 50%. His 24 together with Pro 25 constitute the joint between the NH 2 -terminal helix and helix I of the globular domain. Infrared spectroscopy shows that interaction with the DNA induces an amount of ␣-helical structure equivalent to that observed in TFE. As coulombic interactions are involved in complex formation, it is highly likely in the complexes with DNA that the minimal region with ␣-helical structure is that containing the basic cluster. In chromatin, the high positive charge density of the inducible NH 2 -terminal helical element may contribute to the binding stability of the globular domain.The linker histone H1 has a role in the stabilization of both the nucleosome and chromatin higher order structure. Linker histones contain a globular domain flanked by highly basic amino-and carboxyl-terminal tails (1). The terminal domains have, in general, little structure in solution. The COOH-terminal domain acquires, however, a substantial amount of ␣-helix in the presence of secondary structure inducers such as TFE 1 and NaClO 4 (2), suggesting that binding to DNA could stabilize helical segments in the COOH-terminal domain. It has been shown previously by FTIR spectroscopy that a COOH-terminal peptide of histone H1°becomes fully structured upon interaction with the DNA (3). The structures of a turn and of a helix-turn motif belonging to the COOH-terminal domain have been determined by high resolution NMR in the presence of helix stabilizers (4, 5).It is currently accepted that H1 could have a regulatory role in transcription through the modulation of chromatin higher order structure. In vitro experiments with reconstituted chromatin have shown that H1 can repress promotors containing the RNA start site in the linker DNA, and that some sequencespecific transcription factors can counteract the H1-mediated repression. Preferential binding to scaffold-associated regions and participation in nucleosome positioning have been proposed as other possible mechanisms by which H1 could contribute to transcriptional regulation (6 -9). The involvement of H1 in the 300-Å chromatin fiber, which presumably limits the access of the transcriptional machinery, led to the proposal that H1 subtypes may function as generalized repressors. More recen...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.