Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate. A Fourier-transform infrared spectroscopic study

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2(+)-ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.     

Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study.

The helical order parameter of the 26-residue amphiphilic bee venom peptide melittin was measured by polarized attenuated total reflection infrared spectroscopy (ATR-IR) in dry phospholipid multibilayers (MBLs) and when bound to single supported planar bilayers (SPBs) under D2O. Melittin adopted an alpha-helical conformation in MBLs of dipalmitoyl-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), a 4:1 mixture of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and when bound to SPBs of POPC:POPG (4:1). The order parameter of the alpha-helix in the bilayers depended mainly on the type of membrane preparation, and only little on the phospholipid composition of the bilayers. On hydrated SPBs, the helical order parameter was negative, indicating that the alpha-helix long axis of melittin was preferentially oriented parallel to the plane of the supported membrane. However, in dry MBLs, the helical order parameter was positive, indicating that the alpha-helix of melittin was preferentially oriented parallel to the phospholipid fatty acyl chains. It is concluded that the orientation of melittin in membranes depends on the degree of hydration of the model membranes rather than on the technique which is used for its determination. ATR-IR spectroscopy of polypeptides in or associated with supported planar membranes in D2O may become a useful tool for the determination of their orientation in and on membranes.     

A high-pressure, infrared spectroscopic study of the solvation of bilirubin in lipid bilayers

The location of bilirubin IXa in lipid bilayers of dimyristoylphosphatidylcholine or dioleoylphosphatidylcholine was studied by determining the effects of bilirubin on the infrared spectra of the lipids as a function of pressure. It was found for both bilayers that bilirubin intercalated into the polymethylene chain region of the bilayer, being located between the carbonyl region and the methylene group two carbons from the methyl terminus. Small amounts of bilirubin interacted with the choline region of dioleoylphosphatidylcholine. Lesser amounts interacted with the choline region of dimyristoylphosphatidylcholine. This difference between the two types of bilayers was attributed to the degradation of small amounts of bilirubin IXa to more polar isomers in the presence of dioleoylphosphatidylcholine. In dioleoyl- but not dimyristoylphosphatidylcholine, bilirubin interacted with the C = O region, probably indicating that bilirubin in the latter type of bilayer was intercalated into the polymethylene chains above and below the double bond. Bilirubin decreased the pressure required for the liquid-crystal to gel-phase transition in both bilayers at 28 degrees C. Bilirubin was not forced out of either bilayer at pressures as high as 20 kbar.     

A Fourier-transform infrared spectroscopic study of the phosphoserine residues in hen egg phosvitin and ovalbumin

A Fourier-transform infrared spectroscopic study of hen egg phosvitin and ovalbumin has been carried out. Bands arising from monoanionic and dianionic phosphate monoester [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498] can be identified easily in the 1300-930 cm-1 region in spectra of solutions of O-phosphoserine and phosvitin, a highly phosphorylated protein. On the other hand, spectra of ovalbumin show a relatively strong absorption above 1000 cm-1 arising from the protein moiety. Below 1000 cm-1, a single band at 979 cm-1 is observed; this band is not present in spectra of dephosphorylated ovalbumin, and therefore, it has been assigned to the symmetric stretching of the phosphorylated Ser-68 and Ser-344 in the dianionic ionization state. In addition, bands arising from symmetric and antisymmetric stretchings of the monoanionic ionization state, and from the antisymmetric stretching of the dianionic state, can be detected above 1000 cm-1 in difference spectra of ovalbumin minus dephosphorylated ovalbumin. The effect of pH on the infrared spectra of O-phosphoserine, phosvitin, and ovalbumin is consistent with the phosphoserine residues undergoing ionization with pK values about 6. This study demonstrates that Fourier-transform infrared spectroscopy can be a useful technique to assess the ionization state of phosphoserine residues in proteins in solution.     

Effects of cis and trans unsaturation on the structure of phospholipid bilayers: a high-pressure infrared spectroscopic study

In order to compare the effects of cis and trans unsaturation on the structure and packing of phospholipid bilayers, infrared spectra of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) were measured in a diamond anvil cell at 28 degrees C as a function of pressure up to 36 kbar. The infrared spectra indicate that DEPC and DOPC undergo pressure-induced liquid-crystalline to gel phase transitions at critical pressures of 0.7 and 5.2 kbar, respectively. Below their respective critical pressures, the infrared spectra of DOPC and DEPC are essentially indistinguishable, whereas above these pressures, there are very pronounced differences in the barotropic behavior of these two lipids. Specifically, at the 5.2-kbar transition in DOPC, there are significant changes in the frequencies, intensities, and widths of bands associated with the interfacial C = O groups, the olefinic CH = CH groups, and the terminal CH3 groups, whereas the corresponding bands of DEPC are, by contrast, relatively insensitive to the pressure-induced phase transition. The unusual band shape changes in DOPC are attributed to a unique packing arrangement of the oleoyl acyl chains required to accommodate the bent geometries of adjacent cis double bonds. Moreover, above 5 kbar in DEPC, well-defined correlation field splittings of the CH2 scissoring and rocking modes are observed, with magnitudes very similar to those observed at comparable pressures in saturated lipid systems. The absence of correlation field splittings of the corresponding bands of DOPC up to 36 kbar suggests that the bent oleoyl acyl chains are closely packed with all chains oriented parallel to each other.     

Orientation of gramicidin D incorporated into phospholipid multibilayers: a Fourier transform infrared-attenuated total reflection spectroscopic study

Polarized Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy was applied to study the orientation of the linear pentadecapeptide antibiotic gramicidin D incorporated into phospholipid multibilayers, which were cast on a germanium ATR plate from chloroform solution. In DMPC and DPPC multibilayers, the CH2 stretching bands of lipid hydrocarbon chains were slightly shifted to the higher frequency side and bandwidth was increased in the presence of gramicidin. However, in DPPE multibilayers, frequencies and bandwidths of these bands were unaltered. In each case, gramicidin produced little effect on the orientation of lipid hydrocarbon chains, suggesting that gramicidin penetrates into lipid layers without noticeable perturbations. Upon incubation of cast films in contact with water above the gel-liquid-crystalline transition temperature (Tc) of lipids, the reorientation of gramicidin in lipid multibilayers occurred, the degree thereof depending upon the fluidity of the lipid hydrocarbon chains and the amount of surrounding water. In DMPC multibilayers, the helix axis of gramicidin was oriented almost parallel to the lipid hydrocarbon chains after incubation. In DPPC multibilayers, on the other hand, the helix axis of gramicidin was tilted on average about 15 degrees from the lipid hydrocarbon chains after incubation. However, in DPPE multibilayers, which are known to have the most rigid bilayer structures, the reorientation of gramicidin could not be seen.     

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.     

The protein environment surrounding tyrosyl radicals D. and Z. in photosystem II: a difference Fourier-transform infrared spectroscopic study.

Photosystem II contains two redox-active tyrosine residues, termed D and Z, which have different midpoint potentials and oxidation/reduction kinetics. To understand the functional properties of redox-active tyrosines, we report a difference Fourier-transform infrared (FT-IR) spectroscopic study of these species. Vibrational spectra associated with the oxidation of each tyrosine residue are acquired; electron paramagnetic resonance (EPR) and fluorescence experiments demonstrate that there is no detectable contribution of Q(A)- to these spectra. Vibrational lines are assigned to the radicals by isotopic labeling of tyrosine. Global 15N labeling, 2H exchange, and changes in pH identify differences in the reversible interactions of the two redox-active tyrosines with N-containing, titratable amino acid side chains in their environments. To identify the amino acid residue that contributes to the spectrum of D, mutations at His189 in the D2 polypeptide were examined. Mutations at this site result in substantial changes in the spectrum of tyrosine D. Previously, mutations at the analogous histidine, His190 in the D1 polypeptide, were shown to have no significant effect on the FT-IR spectrum of tyrosine Z (Bernard, M. T., et al. 1995. J. Biol. Chem. 270:1589-1594). A disparity in the number of accessible, proton-accepting groups could influence electron transfer rates and energetics and account for functional differences between the two redox-active tyrosines.     

An infrared spectroscopic study of metastable and stable forms of hydrated cerebroside bilayers

Fourier transform infrared spectroscopy has been used to study the stable and metastable forms of a range of cerebrosides in aqueous systems. The spectra provide evidence for different degrees of inter- and intra-molecular hydrogen bonding, involving principally the amide group, in these different states. A comparison has been made with the spectra of a cerebroside containing an alpha-hydroxyl group in the fatty acyl chain. This cerebroside does not show metastability and its hydrogen bonding characteristics are shown to be different.     

Structural analyses of a channel-forming fragment of colicin E1 incorporated into lipid vesicles. Fourier-transform infrared and tryptophan fluorescence studies.

Structural changes upon binding to the membrane of a COOH-terminal channel-forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles.     

Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.     

Influence of voltage and ATP on ion-channel of (Na,K)ATPase incorporated into solvent-free phospholipid planar bilayers

Purified (Na,K)ATPase was incorporated into solvent free phospholipid bilayers made on patch-clamp pipettes. In the absence of ATP, the incorporated enzyme acted as an ion-channel which underwent opening and closing (switching) upon application of transmembrane potential gradient of more than 40 mV. The minimum conductance was about 40 pS. It was inhibited by ouabain from one side. ATP added to the opposite side shifted the threshold potential for switching of the channel to 80 mV. Furthermore the magnitude of minimum conductance decreased to 6-10 pS in the presence of ATP.     

Electrochemical modeling of electron and proton transfer to ubiquinone-10 in a self-assembled phospholipid monolayer.

Ubiquinone-10 (UQ) was incorporated at concentrations ranging from 0.5 to 2 mol% in a self-assembled monolayer of dioleoylphosphatidylcholine (DOPC) deposited on a mercury drop electrode, and its electroreduction to ubiquinol (UQH2) was investigated in phosphate and borate buffers over the pH range from 7 to 9.5 by a computerized chronocoulometric technique. The dependence of the applied potential for a constant value of the faradaic charge due to UQ reduction upon the electrolysis time t at constant pH and upon pH at constant t was examined on the basis of a general kinetion approach. This permitted us to conclude that the reduction of UQ to UQH2 in DOPC monolayers takes place via the reversible uptake of one electron with the formation of the semiubiquinone radical anion UQ.-, followed by the rate-determining protonation of this anion with UQH. formation; this neutral radical is more easily reduced than UQ, yielding the ubiquinol UQH2. In spite of the very low concentration of hydrogen ions as compared with that of the acidic component of the buffer, the only effective proton donor is the proton itself; this strongly suggests that the protonation step takes place inside the polar head region of the DOPC monolayer, which is only accessible to protons.     

Voltage dependence of liver gap-junction channels reconstituted into liposomes and incorporated into planar bilayers.

The voltage dependence of rat liver gap junctions was investigated using non-denaturing solubilization and reconstitution of gap-junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single-channel conductance was voltage independent, and its magnitude was 700-1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed-state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage-sensitive conformational changes.     

Secondary structure of the membrane-bound form of the pore-forming domain of colicin A. An attenuated total-reflection polarized Fourier-transform infrared spectroscopy study.

The structure of the pore-forming domain of the bacterial toxin colicin A was studied by attenuated total-reflection polarized Fourier-transform infrared spectroscopy. This channel-forming fragment interacts with dimyristoylglycerophosphoglycerol (Myr2GroPGro) vesicles and forms disk-like complexes. Analysis of the shape of the amide I' band indicates that its secondary structure is not affected by the pH 5.0-7.2. However, 5-10% of the peptide amino acids adopt an alpha-helical structure upon complex formation with Myr2GroPGro, while the random-coil and beta-sheet structure contents decrease. Interestingly, the increase in alpha-helical content is essentially due to an increase in the high-frequency component of the alpha-helical domain of amide I'. The fact that only this component was 90 degrees polarized (i.e. the helix is parallel to the acyl chain) suggests that only this particular type of helix is associated with the Myr2GroPGro bilayer.     

The interfacial structure of phospholipid bilayers: differential scanning calorimetry and Fourier transform infrared spectroscopic studies of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine and its dialkyl and acyl-alkyl analogs.

The thermotropic phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) and its 1,2-dialkyl, 1-acyl 2-alkyl and 1-alkyl 2-acyl analogs was examined by differential scanning calorimetry, and the organization of these molecules in those hydrated bilayers was studied by Fourier transform infrared spectroscopy. The calorimetric data indicate that substitution of either or both of the acyl chains of DPPC with the corresponding ether-linked hydrocarbon chain results in relatively small increases in the temperature (< 4 degrees C) and enthalpy (< 1 kcal/mol) of the lipid chain-melting phase transition. The spectroscopic data reveal that replacement of one or both of the ester-linked hydrocarbon chains of DPPC with its ether-linked analog causes structural changes in the bilayer assembly, which result in an increase in the polarity of the local environments of the phosphate headgroups and of the ester carbonyl groups at the bilayer polar/apolar interface. The latter observation is unexpected, given that ester linkages are considered to be intrinsically more polar that ether linkages. This finding cannot be satisfactorily rationalized unless the conformation of the glycerol backbones of the analogs containing ether-linked hydrocarbon chains differs significantly from that of diacyl glycerolipids such as DPPC. A comparison of the alpha-methylene scissoring bands and the methylene wagging band progressions of these lipids with the corresponding absorption bands of specifically chain-perdeuterated analogs of DPPC also supports the conclusion that replacement of the ester-linked hydrocarbon chains of DPPC with the corresponding ether-linked analog induces conformational changes in the lipid glycerol backbone. The suggestion that the conformation of glycerol backbones in the alkyl-acyl and dialkyl derivatives of DPPC differs from that of the naturally occurring 1,2-diacyl glycerolipid suggests that mono- and di-alkyl glycerolipids may not be good models of their diacyl analogs. These results, and previously published evidence that DPPC analogs with ether-linked hydrocarbon chains spontaneously form chain-interdigitated gel phases at low temperatures, clearly indicate that the properties of lipid bilayers can be substantially altered by small changes in the chemical structures of their polar/polar interfaces, and highlight the critical role of the interfacial region as a determinant of the structure and organization of lipid assemblies.     

Interaction of substance P with phospholipid bilayers: A neutron diffraction study.

Neutron diffraction has been used to study the membrane-bound structure of substance P (SP), a member of the tachykinin family of neuropeptides. The depth of penetration of its C-terminus in zwitterionic and anionic phospholipid bilayers was probed by specific deuteration of leucine 10, the penultimate amino acid residue. The results show that the interaction of SP with bilayers, composed of either dioleoylphosphatidylcholine (DOPC), or a 50:50 mixture of DOPC and the anionic phospholipid dioleoylphosphatidylglycerol (DOPG), takes place at two locations. One requires insertion of the peptide into the hydrophobic region of the bilayer, the other is much more peripheral. The penetration of the peptide into the hydrophobic region of the bilayer is reflected in a marked difference in the water distribution profiles. SP is seen to insert into DOPC bilayers, but a larger proportion of the peptide is found at the surface when compared to the anionic bilayers. The positions of the two label populations show only minor differences between the two types of bilayer.     

A calorimetric and infrared spectroscopic study of the stabilizing solute proline.

We have studied the calorimetric and infrared spectroscopic properties of the amino acid proline which has been implicated in the stabilization of biomacromolecules during reduced water states. It has been suggested that the ability of this molecule to protect biomacromolecules during these stress states may be related to the formation of polymeric aggregates of proline monomers in solution. The structure of this aggregate is thought to be an alternates stack, forming a hydrophilic colloid-like polymer which is thought to interact with hydrophobic moieties of biomacromolecules, reducing the exposed hydrophobic area during reduced water conditions. Calorimetric data presented in this work show that in increasing concentration of proline in solution the enthalpy associated with the melting of bulk water is greatly reduced, indicating strong hydrogen bonding character of proline in aqueous solution. Proline shows two eutectic phase separations at moderate concentrations and one of these eutectics may be the proposed intermolecular state. A partial phase diagram for proline is presented. Fourier-transform infrared spectroscopic data indicate that the COO- asymmetric stretch of proline shows marked splitting with increasing proline concentration. This suggests that the carboxylate is in different environments, with the high energy vibrations representing COO- groups which are participating in the hydrogen bonding pattern associated with the formation of the intermolecular stack. Changes in the CH2 asymmetric and symmetric stretches of the pyrrolidine rings of proline are consistent with the proposed stack structure. We also suggest a possible mechanism by which these intermolecular associations may be important in the protection of biomacromolecules during reduced water states.