Chain motions in lipid-water and protein-lipid-water phases: a spin-label and x-ray diffraction study

An attempt has been made to establish a relation between the rapid reorientational motions of the lipid chains and the structure of the lipid-water and the protein-lipid-water phases. The movements were studied by electron paramagnetic resonance spectra of the fatty acids labelled at different positions along the chain; the structure of the phases, established by X-ray diffraction, differs by the long-range organization (lamellar, hexagonal, etc.) and by the short-range conformation of the chains (liquid-like, α; stiff and hexagonally packed, β or β′). The rapid motions of the hydrocarbon chains are found to be largely independent of the long-range order and highly dependent on the short-range conformation. The mobility of the nitroxide group incorporated within regions in the β (or β′) conformation is low and independent of its position along the chain. On the contrary, the mobility of the nitroxide group incorporated in regions in the α-conformation is high and increases as the position is moved along the chain away from the polar end. Temperature and water content affect the motions of the chains in the α but not in the β-conformation. Moreover, the labels appear to be perfectly soluble in the α regions, whereas the solubility in the βregions varies with the position of the nitroxide group and with the nature of the lipid. As a consequence, among the numerous phase transitions observed in these systems only those which involve a change in the conformation of the chains are accompanied by profound perturbations of the spin-label spectra; when the label is soluble in the β-regions, a discontinuity in chain motions is observed at the α-β transition; when the label is less soluble in the β than in the α-regions, its concentration in the α-regions increases as the α-β transition proceeds and magnetic interactions can be observed. In lipoprotein phases with electrostatic interactions the motions of the chains appear to be unperturbed by the presence of the protein. If the interactions are of the hydrophobic type, the electron paramagnetic resonance spectra indicate that some regions of the chains come in contact with the proteins.     

Spectroscopic characterization of poly(Glu-Ala)

Infrared linear dichroism and ultraviolet circular dichroism speetroscopy have been used to distinguish four conformational forms of the ionizable sequential polypeptide poly(Glu-Ala). Two of these conformations, the α helix and the β form, were observed for the unionized polypeptide in solution. The α helix appeared immediately upon neutralization of the side-chain carboxyl functions, whereas the β form was observed after the neutralized solution had been standing for several days. The β form was also observed for films cast from either high or low pH solutions. Ionization of the glutamyl residues resulted in a circular dichroism spectrum which has previously been observed for charged homopolymers and appears to result from an extended helical conformation. Further, heating either the α helical or the charged extended helix resulted in a transition to a disordered chain. These results are consistent with the results of conformational calculations presented elsewhere.     

Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases

This work describes the structure of a variety of lecithin-water phases observed below the “melting” temperature of the hydrocarbon chains, with special emphasis on the conformation of the chains. The lecithins studied in this work are the homologous series dioctanoyl to distearoyl, 2-decanoyl-1-stearoyl, and a preparation from hen eggs. The hydrocarbon chains are found to adopt a variety of conformations in addition to type α, the liquid-like organization observed above the melting temperature. Type β: the chains are stiff and parallel, oriented at right angles to the plane of the lamellae and packed with rotational disorder in a two-dimensional hexagonal lattice ($\text{a ~ 4.85}\text{A}\text{?}\text{)}$. Type β′: similar to β, but with the chains tilted with respect to the normal to the lamellae. Type δ: the chains are probably coiled into helices, whose axes are perpendicular to the plane of the polar groups and are packed with rotational disorder in a two-dimensional square lattice ($\text{a ~ 4.80}\text{A}\text{?}\text{)}$, α is the predominant conformation, common to most lipids in the presence of water and at sufficiently high temperature, and the one more relevant to membranes; β is observed at lower temperatures in lipids whose chains are heterogeneous and in the presence of very small amounts of water; β′ is found in synthetic lecithins with identical chains, in the presence of variable amounts of water; δ is observed in dry lecithins. A highly ordered crystalline phase, yet displaying rotational disorder of the chains, is observed in almost dry lecithins. Most of the phases are lamellar, and contain one lipid bilayer per repeat unit. Two phases display two-dimensional lattices: Pδ, formed by ribbon-like elements with the chains in the δ conformation; Pβ′, formed by lamellae of type β′ distorted by periodic ripples. The results emphasize the clear-cut difference between the liquid-like and the other types of partly ordered conformations, as well as the correlations which exist between the chemical composition and the structure of the lipids below the melting temperature of the chains.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Conformation of poly-L-tyrosine in aqueous solution

The conformational transition of poly-L -tyrosine in 0.1M KCl was investigated by ORD and infrared spectroscopy, potentiometric titration, and sedimentation velocity experiments. It is shown that the fully ordered conformer is obtained by slow titration of the random coil with 0.1N HCl at 25°C. The charge-induced transition, at variance with other poly-α-amino acids, is completed in a narrow range of α. An aggregation process was detected both by potentiometric titration and sedimentation velocity. The polyamino acid aggregates around α = 0.7 at 25°C when the conformational transition is almost complete. Infrared spectra, in the region of the amide I band (1650 cm^?1) showed that the transition is a random coil → antiparallel β one. Evidence exists that the form is of the intramolecular type. The foregoing interpretations of ORD and CD spectra in terms of the α-helix conformation are discussed.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain.

The phases of simple systems involving one type of protein (lysozyme or cytochrome c) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperature than the one observed in the case of the phase with electrostatic interaction.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain

The phases of simple systems involving one type of protein (lysozyme or cytochrome $\text{c}$) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperture than the one observed in the case of the phase with electrostatic interaction.     

Lipid-water interface mediates reversible ionophore conformational change

A new procedure of conformational analysis was used to demonstrate that the ionophore conformation is mediated by its membrane environment. In the hydrophobic lipid matrix, the ionomycin-Ca++ complex adopts a conformation well suited for translocation across the interior of the membrane whereas at the lipid-water interface, the Ca++ ion is immersed into the aqueous phase in a position favorable to its complexation or decomplexation. The translocation of Ca++ across the lipid bilayer supposes a reversible transformation of the two conformers. The conformational analysis shows how the dielectric constant discontinuity existing at the lipid-water interface mediates the reversible transformation of one structure into the other.     

Conformation of oligopeptides with L-leucyl-L-leucyl-L-alanyl and L-methionyl-L-methionyl-L-leucyl repeating units

The conformation of oligopeptides with hydrophobic side chains, Nps-(L -Leu-L -Leu-L -Ala)_n-OEt and Nps-(L -Met-L -Met-L -Leu)_n-OEt(n = 1–6), in the solid state, obtained either by evaporation of the solvent or by precipitation with diethyl ether from a 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solution, has been studied with ir spectroscopy and x-ray powder-diffraction measurements. The conformation of these peptides in the HFIP solution has been studied by CD spectroscopy. Due to a strong preference of the amino acids to form an α helix, the peptides begin forming α helices at the dodecapeptide in the HFIP solution, and in the solid state by evaporation. In the solid state, with precipitation, the α-helical conformation is first observed at the octadecapeptide and the lower peptides assume a β structure. The conformational change, from the α helix to the β structure of the peptides with 12 to 15 amino acid residues, during the precipitation process, is due to a strong tendency of the amino acids to form the β-structure in rather short peptide lengths.     

Laser Raman studies of conformational variations of poly-L-lysine

The frequencies and intensities of the laser Raman spectra of poly-L -lysine (PLL) have been observed in the following studies: (1) the thermally induced α-to-β transition which occurs with increasing temperature at high pH; (2) the ionized form to α transition at 10°C by increasing pH; and (3) the ionized form to α transition by ionic strength at low pH. The frequency-dependent bands which have been observed are the amide I (in H₂O), amide I′ (in D₂O), amide III, and C–C stretch. It has been found possible to assign an unique set of frequencies and intensities to each conformation of PLL of α, β, and ionized form. In this way the nature of the conformations intermediate in the transitions can be determined. The frequencies of the amide III and amide III′ are very weak in the α-helix and somewhat higher than usual in the β form. Hence it appears the amide III and amide III′ bands may differ from one type of polypeptide to another with the same backbone conformation.     

Azobenzene-Containing polypeptides: Photoregulation of conformation in solution

Photochromic polypeptides, with 16 to 56% azobenzene groups in the side chains, have been prepared by reaction of poly(L -glutamic acid) with p-aminozaobenzene, both in the presence of dicyclohexyl carbodiimide/N-hydroxybenzotriazole and of pivaloyl chloride. Analogous modification reactions carried out on poly(L -aspartic acid) were unsuccessful owing to the formation of N-succinimide rings. In trimethylphosphate, all the azopolypeptides exhibit the α-helix CD pattern. Irradiation produces the trans-to-cis isomerization of the azo side chains, but does not induce any variations of the backbone conformation. In water, the CD spectra indicate the presence of appreciable amounts of α helix in 16 and 21% mol azo-containing poly-(L glutamates), while a β structure is present in a 36% mol azopolypeptide. Light produces conformational changes of the polypeptide conformation which are completely reversed in the dark. The extent and kind of photobehavior depend on the azo content and the pH value at which irradiation is carried out. The light-induced effects are discussed on the basis of the pH-induced order-disorder conformational transitions. In fact, the pK values and the transition curves of the dark-adapted samples were found to be different from those of the irradiated ones.     

Tilted hydrocarbon chains of dipalmitoyl lecithin become perpendicular to the bilayer before melting.

Differential scanning calorimetry studies of dipalmitoyl lecithin show two reversible transitions as the temperature is changed between 20 and 50 degrees C. A pretransition endotherm occurs at 35 degrees C prior to the main chain melting endotherm which occurs at 42 degrees C. X-ray diffraction studies show that below 33 degrees C the chains of the lecithin are fully extended, packed in a hexagonal crystalline lattice but tilted with respect to the plane of the bilayer. Between 35 and 42 degrees C the chains are similarly packed but oriented perpendicular to the bilayer plane. Above 44 degrees C the chains are "melted" or disordered. Monolayer studies of dipalmitoyl lecithin using continuous recording of pressure with molecular area reveal the existence of two solid condensed phases corresponding to these tilted and verticle chain structures. The tilted to perpendicular transition would account for the pretransition endotherm of the lipid; the crystalline to melted change corresponds to the larger transition observed at 42 degrees C.     

Symmetry distortion in the human hemoglobin tetramer induced by asymmetric ligation

To investigate the conformational changes in human tetrameric (αβ)(2) hemoglobin upon binding of the first two ligands, we have measured the kinetics of reactions between 4,4'-dithiodipyridine and β93Cys sulfhydryl groups of four diliganded hemoglobins by using CO-bound Fe(II)-Ni(II) hybrids with and without β-β cross-linking. The data show that all the diliganded intermediates have high sulfhydryl reactivities, which are greater than or equal to that for the fully-liganded end state, especially when containing liganded α subunit(s). The results also reveal that both the asymmetrically (α1β1 and α1β2) diliganded species show similar high rates of sulfhydryl reactivity and biphasic kinetics, suggesting a new conformation but only slight functional distortion caused by asymmetric ligation.     

The two beta forms of poly(L-glutamic acid).

β-Helical poly(L -glutamic acid) in a gel state was found to be easily converted to the antiparallel β form by heating. Two β forms were obtained, depending on the temperature of heating. Temperatures between 40° and 85°C produced a β form with a spacing between pleated sheets (d₀₀₁) of 9.03 Å, termed β₁. If the heating was carried out at temperatures higher than 85°C, the β₁ form underwent another conformational transition reducing the d₀₀₁ value from 9.03 to 7.83 Å (termed β₂) without any prominent change in the fiber repeat distance (i.e., the polypeptide backbone conformation). The time course of these two transitions was followed by measuring the infrared spectra of the samples, and it was concluded that the α → β₁ transition in its initial stage obeys a pseudo-first order rate process with activation enthalpy and entropy of 54 kcal/mol and 92 eu, respectively. On the other hand, the typical sigmoidal conversion curves observed for the transition between the two types of β forms (β₁ → β₂) indicate that this transition proceeds via a socalled “nucleation and growth” process. The kinetic theory of phase transitions developed by Avrami can be applied with success to explain this transition. The infrared spectra, in the region from 1800 to 200 cm^?1, were measured for these two β forms and the results showed that the conformation of the side chains and the mode of the hydrogen bonding between the side-chain carboxyl groups undergo appreciable change during the transition. The heat-induced conformational transition of poly(L -Glu⁷⁸ L -Val²²) was also studied. The copolymer was transformed from the α-helical conformation directly to the β₂ form. The reason for this was thought to be due to the fact that the L -valine residues and the L -glutamyl residues near the L -valine residues have a strong tendency to take the more compact β₂ form.     

THE STRUCTURE OF THE LIQUID-CRYSTALLINE PHASES OF LIPID-WATER SYSTEMS

Some simple lipid-water systems have been studied by x-ray scattering techniques, as a function of lipid concentration and temperature. Several liquid-crystalline phases have been found, and their structure has been determined: only one of these is lamellar. In all these phases the hydrocarbon part of the lipid molecules has a disordered, liquid-like structure. One biological phospholipid, a human brain extract, has been studied by the same technique, and two liquid-crystalline phases have been found: a lamellar phase, built up by an ordered sequence of lipid and water planar sheets, and a hexagonal phase, which is a hexagonal array of circular cylinders, each cylinder being a thin water channel covered by the hydrophilic groups of the lipid molecules, the hydrocarbon chains filling the gap between the cylinders. The interpretation of the electron microscope observations of the structure of lipoprotein membranes is discussed, and some possible biological implications are suggested.     

Single crystals of poly(L-glutamic acid)

Lamellar single crystals of alkaline earth salts of poly(L -glutamic acid) have been grown by precipitation from dilute aqueous solution and studied by optical and electron microscopy and by x-ray and electron diffraction. The calcium, strontium and barium salts were crystallized in the β form above room temperature and could be converted to crystals of β-poly(L -glutamic acid) by washing in dilute hydrochloric acid. The magnesium salt, on the other hand, was crystallized in the α form at or below room temperature but could not be converted into crystals of α-poly(L -glutamic acid) by washing in hydrochloric acid. The crystalline lamellae are very thin (thicknesses range from 25 to 60 Å in β crystals and are about 100 Å in α crystals) and the polypeptide chains are oriented normal to the planes of the lamellae. It is clear from the disparity between crystal thickness and molecular length that the molecules crystallize by folding at the upper and lower surfaces of the crystals. Conformations of the molecules at these folds are discussed briefly.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Diacetylenic lipid microstructures: structural characterization by X-ray diffraction and comparison with the saturated phosphatidylcholine analogue

Thermotropic and lyotropic mesomorphism in the polymerizable lecithin 1,2-ditricosa-10,12-diynoyl-sn-glycero-3-phosphocholine and its saturated analogue, 1,2-ditricosanoyl-sn-glycero-3-phosphocholine, has been investigated by wide- and low-angle X-ray diffraction of both powder and oriented samples and by differential scanning calorimetry. Previous studies have shown that the hydrated diacetylenic lipid forms novel microstructures (tubules and stacked bilayer sheets) in its low-temperature phase. The diffraction results indicate that at low temperatures fully hydrated tubules and sheets have an identical lamellar repeat size (d001 = 66.4 A) and crystalline-like packing of the acyl chains. Chain packing in the lamellar crystalline phase is hydration independent. A model for the polymerizable lecithin with (1) fully extended all-trans methylene segments, (2) a long-axis tilt of 32 degrees, and (3) minimal chain interdigitation seems most reasonable on energetic grounds, is consistent with the diffraction data (to 3.93-A resolution), and is likely to support facile polymerization. Above the chain "melting" transition the lamellar repeat of the polymerizable lipid increases to 74 A. The conformational similarity between tubules, sheets, and the dry powder is corroborated by calorimetry, which reveals a cooling exotherm at the same temperature where tubules form upon cooling hydrated sheets. The data suggest that although a high degree of conformational order is a pertinent feature of tubules, this character alone is not sufficient to account for tubule formation. The conformation of the corresponding saturated phosphatidylcholine appears to be similar to that of other saturated phosphatidylcholines in the lamellar gel phase. Furthermore, above the main transition temperature, the dry, saturated lipid shows evidence of a P delta phase (112 degrees C), whereas the diacetylenic lipid appears to exhibit a centered rectangular phase, R alpha (55 degrees C).