Refinement of Singer-Nicolson fluid-mosaic model by microscopy imaging: Lipid rafts and actin-induced membrane compartmentalization

This year celebrates the 50th anniversary of the Singer-Nicolson fluid mosaic model for biological membranes. The next level of sophistication we have achieved for understanding plasma membrane (PM) structures, dynamics, and functions during these 50 years includes the PM interactions with cortical actin filaments and the partial demixing of membrane constituent molecules in the PM, particularly raft domains. Here, first, we summarize our current knowledge of these two structures and emphasize that they are interrelated. Second, we review the structure, molecular dynamics, and function of raft domains, with main focuses on raftophilic glycosylphosphatidylinositol-anchored proteins (GPI-APs) and their signal transduction mechanisms. We pay special attention to the results obtained by single-molecule imaging techniques and other advanced microscopy methods. We also clarify the limitations of present optical microscopy methods for visualizing raft domains, but emphasize that single-molecule imaging techniques can “detect” raft domains associated with molecules of interest in the PM.     

Refinement of the structure of beta-chitin.

The structure of β-chitin has been refined by rigid-body least-squares methods, based on the intensity data for highly crystalline specimens from the pogonophore Oligobrachia ivanovi. The structure consists of an array of poly-N-acetyl-D -glucosamine chains all having the same sense, which are linked together in sheets by N? H … O?C hydrogen bonding of the amide groups. In addition to the O-3′? H … O-5 intramolecular hydrogen bond, analogous to that in cellulose, the CH₂OH side chain forms an intrasheet hydrogen bond to the carbonyl oxygen on the next chain. This structure shows considerably better agreement between observed and calculated intensities than that possessing an intersheet hydrogen bond, as had been proposed previously. The structure is consistent with the swelling properties of β-chitin and can also be seen to be analogous to that of native cellulose.     

A structure model for the lecithin-cholesterol-water membrane.

Starting from the structural properties of liquid water and making use of the known bond lengths and orientations of the hydrogen-bonded water molecules, models for the water-lecithin and the water-lecithin-cholesterol systems have been derived. The models are in good agreement with the known properties of the basic cell membrane.     

Refinement of the structure of bovine seminal ribonuclease

We report here the refinement at 2.5-Å resolution of the x-ray crystal structure of bovine seminal ribonuclease, a dimeric covalent enzyme. The protein, which crystallizes with one molecule in the asymmetric unit, consists of two subunits of identical chemical sequences, related by an almost exact binary axis. The tertiary structure of the subunits is similar to that of the pancreatic enzyme, which shows similar catalytic properties. The refinement was carried out using the restrained least-squares procedure both in the reciprocal and real spaces. The assemblage of the subunits in the dimer is described and discussed.     

A quantitative model of the domain structure of the photosynthetic membrane

A model is presented that gives a quantitative picture of the distribution of the photosynthetic components in the photosynthetic membrane of higher plants. A salient feature of the model is that most of the pigments are located in the grana where photosystem I and II carry out linear electron transport, whereas the stroma lamellae, which harbour <20% of the pigments, carry out photosystem-I-mediated cyclic electron transport. This arrangement derives from the observation that more pigments are associated with photosystem I, which therefore captures more quanta than photosystem II. The excess pigments associated with photosystem I are thought to be located in the stroma lamellae.     

A modified liquid-mosaic model of plasma membrane structure

A modified liquid-mosaic model of molecular organization of the plasma membrane is proposed, which allow one to interpret non-receptor effects of regulatory peptides and the efficiency of biologically active compounds in extra low concentrations. An evolutionarily earlier type of intercellular chemical signalling (without the participation of receptor structures) is suggested.     

The structure of a cytochrome oxidase-lipid model membrane

The structure of “membranous cytochrome oxidase” has been investigated by X-ray diffraction, optical polarization spectroscopy and EPR spectroscopy. These studies indicate that the cytochrome oxidase molecules are oriented asymmetrically in the membrane profile with a significant portion of their mass occurring within the extravesicular surface of the membrane; the oxidase molecultes span the membrane profile; the distribution of the oxidase molecules over the plane of these membranes is non-crystalline; the oxidase molecules contain bundles of α-helical polypeptide chain segments where the average orientation of the helices is normal to the membrane plane; and the average heme orientation within the oxidase molecules is such that the normal to the heme plane lies in the plane of the membrane.     

Refinement of the mouse model of congenital toxoplasmosis

The goals of the present investigation, focusing on the BALB/c mouse model of congenital toxoplasmosis, were: (1) to find a method to determine pregnancy in the mouse. The method has 100% sensitivity and 72% specificity; (2) to test congenital transmission during the chronic stage of toxoplasmosis. This occurred in 2 of 10 mice tested; (3) to investigate the relationship between the infective dose and the rate of congenital transmission. This was not demonstrated for doses of 10(2) to 10(3) bradyzoites and oocysts of Prugniaud, M3 and M7741 strains, with transmission rates of 3 of 8 to 6 of 10 mice inoculated; (4) to determine homologous and heterologous protection. Homologous protection was demonstrated with Prugniaud cysts, and heterologous protection was found between ME-49 and M3 cysts. This finding is consistent with the uniform natural protection against congenital toxoplasmosis seen in immune women and ewes.     

Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis.

The structure of carp muscle calcium-binding parvalbumin has been refined to an overall residual, ($\text{Σ¦F}{}_0\text{− F}{}_{\mathrm{c}}\text{¦}\text{Σ¦F}{}_0\text{¦}$), of 0.25 by a combination of model building and difference Fourier analyses. The atomic positions were allowed to vary up to 0.25 Å from their idealized positions; individual temperature factors ranged from 2 to 150; and 138 solvent peaks were included in this final structure factor calculation. The effects of varying these parameters were analyzed. The treatment of low-order reflections and the influence of solvent have been analyzed in terms of Babinet's principle. These procedures and results are applicable to other protein refinement problems. The errors in co-ordinates are estimated to range from 0.15 Å for the Ca²⁺ ions to 0.30 Å for internal side chain atoms.A van der Waals' radii study indicates that 42% of the crystal volume is occupied by solvent. There are 16 intermolecular contacts. Of the 108 main chain peptide hydrogen atoms, 15 are neither in contact with solvent nor involved in hydrogen bonds. These “lost” hydrogen bonds are considered to be important to the proposed model of function.     

Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.     

The structure of a cytochrome oxidase-lipid model membrane.

The structure of "membranous cytochrome oxidase" has been investigated by X-ray diffraction, optical polarization spectroscopy and EPR spectroscopy. These studies indicate that the cytochrome oxidase molecules are oriented symmetrically in the membrane profile with a significant portion of their mass occurring within the extravesicular surface of the membrane; the oxidase molecultes span the membrane profile; the distribution of the oxidase molecules over the plane of these membranes is non-crystalline; the oxidase molecules contain bundles of alpha-helical polypeptide chain segments where the average orientation of the helices is normal to the membrane plane; and the average heme orientation within the oxidase molecules is such that the normal to the heme plane lies in the plane of the membrane.     

The structure of a model membrane in relation to the viscoelastic properties of the red cell membrane

The molecular arrangement within a lamellar structure composed of human erythrocyte lipids is determined. The 45 A thick lipid layer, in water, is filled in the interior with a liquid-like configuration of the hydrocarbon chains of phospholipid molecules and is covered on both sides by their hydrophilic polar groups. Cholesterol is located so that part of its steroid nucleus is between the polar groups of the phospholipid molecules while the rest of the molecule extends into the inner hydrocarbon layer. This lipid leaflet would be expected to have the mechanical properties of a purely liquid surface, as other authors have shown for the "black" lipid membranes. Data are presented which demonstrate that the intact erythrocyte membrane is a tough viscoelastic substance with a Young''s modulus of 10⁶–10⁸ dynes/cm² and a viscosity of 10⁷–10¹⁰ poises. The parameters and the kinetics of membrane breakdown are incompatible with the model system of pure lipid. Caution must be exercised in applying various data on the model systems to intact membranes.     

Refinement of a cryo-EM structure of hERG: Bridging structure and function

The human-ether-a-go-go-related gene (hERG) encodes the voltage-gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using molecular dynamics flexible fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.     

Refinement of the spatial structure of the gramicidin A ion channel]

The spatial structure of the gramicidin A (GA) transmembrane ion-channel was refined on the base of cross-peak volumes measured in NOESY spectra (mixing time tau m = 100 and 200 ms). The refinement methods included the comparison of experimental cross-peak volumes with those calculated for low-energy GA conformations, dynamic averaging of the low-energy conformation set and restrained energy minimization. Accuracy of the spatial structure determination was estimated by the penalty function Fr defined as a root mean square deviation of interproton distances corresponding to the calculated and experimental cross-peak volumes. As the initial conformation we used the right-handed pi 6,3 LD pi 6,3 LD helix established on the base of NMR data regardless of the cross-peak volumes. The conformation is in a good agreement with NOE cross-peak volumes (Fr 0.2 to 0.5 A depending on NOESY spectrum). For a number of NOEs formed by the side chain protons, distances errors were found as much as 0.5-2.0 A. Restrained energy minimization procedure had little further success. However some of these errors were eliminated by the change in torsional angle chi 2 of D-Leu12 and dynamic averaging of the Val7 side chain conformations. Apparently, majority of deviations of the calculated and experimental cross-peak volumes are due to the intramolecular mobility of GA and cannot be eliminated within the framework of rigid globule model. In summary the spatial structure of GA ion-channel can be thought as a set of low-energy conformations, differing by the side chain torsion angles chi 1 Val7 and chi 2 D-Leu4 and D-Leu10 and the orientation of the C-terminal ethanolamine group. Root mean square differences between the atomic coordinates of conformations are in the range of 0.3-0.8 A.     

HgCl2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.     

Refinement of herpesvirus B-capsid structure on parallel supercomputers.

Electron cryomicroscopy and icosahedral reconstruction are used to obtain the three-dimensional structure of the 1250-A-diameter herpesvirus B-capsid. The centers and orientations of particles in focal pairs of 400-kV, spot-scan micrographs are determined and iteratively refined by common-lines-based local and global refinement procedures. We describe the rationale behind choosing shared-memory multiprocessor computers for executing the global refinement, which is the most computationally intensive step in the reconstruction procedure. This refinement has been implemented on three different shared-memory supercomputers. The speedup and efficiency are evaluated by using test data sets with different numbers of particles and processors. Using this parallel refinement program, we refine the herpesvirus B-capsid from 355-particle images to 13-A resolution. The map shows new structural features and interactions of the protein subunits in the three distinct morphological units: penton, hexon, and triplex of this T = 16 icosahedral particle.     

Refinement of the solution structure of a branched DNA three-way junction.

We have refined the structure of the DNA Three-Way Junction complex, TWJ-TC, described in the companion paper by quantitative analysis of two 2D NOESY spectra (mixing times 60 and 200 ms) obtained in D2O solution. NOESY crosspeak intensities extracted from these spectra were used in two kinds of refinement procedure: 1) distance-restrained energy minimization (EM) and molecular dynamics (MD) and 2) full relaxation matrix back calculation refinement. The global geometry of the refined model is very similar to that of a published, preliminary model (Leontis, 1993). Two of the helical arms of the junction are stacked. These are Helix 1, defined by basepairs S1-G1/S3-C12 through S1-C5/S3-G8 and Helix 2, which comprises basepairs S1-C6/S2-G5 through S1-G10/S2-G1. The third helical arm (Helix 3), comprised of basepairs S2-C6/S3-G5 through S2-C10/S3-G1 extends almost perpendicularly from the axis defined by Helices 1 and 2. The bases S1-C5 and S1-C6 of Strand 1 are continuously stacked across the junction region. The conformation of this strand is close to that of B-form DNA along its entire length, including the S1-C5 to S1-C6 dinucleotide step at the junction. The two unpaired bases S3-T6 and S3-C7 lie outside of the junction along the minor groove of Helix 1 and largely exposed to solvent. Analysis of the refined structure reveals that the glycosidic bond of S3-T6 exists in the syn conformation, allowing the methyl group of this residue to contact the hydrophobic surface of the minor groove of Helix 1, at S3-G11.(ABSTRACT TRUNCATED AT 250 WORDS)     

A model for the structure of fumarate reductase in the cytoplasmic membrane of Escherichia coli

By a recombinant DNA approach we have prepared Escherichia coli cytoplasmic membranes that are highly enriched in the terminal electron transfer enzyme fumarate reductase. This enzyme is composed of four nonidentical subunits in equal molar ratio. A 69,000-dalton covalent flavin-containing subunit and a 27,000-dalton nonheme iron-containing subunit make up a membrane extrinsic catalytic domain. Two very hydrophobic subunits of 15,000 and 13,000 daltons make up the hydrophobic membrane anchor domain. Electron microscopy of negatively stained membranes shows a characteristic knob-and-stalk-type structure composed of the catalytic domain. The anchor polypeptides have been analyzed for hydrophobic segments and alpha-helical content and a model for their organization within the lipid bilayer is presented. The results reviewed in this paper suggest a model for the fumarate reductase complex in the cytoplasmic membrane.