Protein-sodium dodecyl sulfate complexes: determination of molecular weight, size and shape by controlled pore glass chromatography

The peak position vs log molecular weight curves of protein-SDS complexes chromatographed on controlled pore glass of narrow pore size distribution is linear over a molecular weight range of 17,000–385,000. A glass with a pore size of approximately 500 Å allows the inclusion of all complexes in this range. Peak position curves on glasses with broad pore distributions show decreased resolution and deviate from linearity at low elution coefficients.Exclusion size analysis of the elution coefficients of individual complexes from different columns with pore diameters ranging from 197 to 650 Å gives from 120 to 423 Å as their longest dimension. Assuming constant hydration and SDS-to-protein ration, the found dimension suggests the shape of a football, rather than a sphere or rigid rod.     

X-Ray Characterization of Melanins—I

The intrinsic local structure characterization of natural sepia melanin and L-dopa and tyrosine synthetic melanin powder has been carried out by X-ray diffraction using synchrotron radiation. The derived structure factor, S(q), shows six significant diffuse peaks within the q-range from 0.3 Å^-1 to 16 Å^-1 in the reciprocal space (q= (4π sin θ)/λ, 2θ is the scattering angle). The Fourier transform of S(q), which yields the radial distribution function (RDF), gives us information in real space of a 1.42 Å distance averaged over the C-C, C-O and C-N bond lengths as well as peaks at 2.40-2.41 Å, 3.67-3.71 Å and 4.67-4.70 Å discrete neighbor distances. There is a great similarity in the scattering intensity profiles of the natural and synthetic melanins indicating that the synthetically prepared material may be essentially similar to “real” melanin in its local atomic arrangements. An evidence of a prepeak at q = 0.45 Å^-1 has been confirmed which indicates a preferred length scale of ～ 13-20 Å that corresponds to the initial particle size in colloidal melanin solutions.     

Small spermatophore size and reduced female fitness in an isolated butterfly population

1. The Glanville fritillary butterfly (Melitaea cinxia L.) has a small population (N_e ～ 100) on the small island of Pikku Tytärsaari (PT) in the Gulf of Finland. The population has remained completely isolated for ～100 generations, which has resulted in greatly reduced genetic variation and high genetic load (low fitness). In particular, females lay small egg clutches with a low egg‐hatching rate in comparison with a large reference population in the Åland Islands (ÅL). 2. In the present study, to what extent egg clutch size and egg‐hatching rate are influenced by male population and spermatophore size was analysed. 3. Spermatophore size increases with male body size, is smaller after the first mating, and is smaller in the small PT population. In the ÅL population but not in the PT population, the egg‐hatching rate increases with spermatophore size. The egg‐hatching rate of PT females is higher when mated with ÅL males than when mated with PT males (heterosis), but there is no such effect on clutch size. The clutch size of ÅL females is, however, reduced when mated with PT males. 4. These results indicate that both male and female traits contribute to reduced reproductive fitness in the small isolated population.     

Surface electric potential on rod-like polyelectrolyte

The electric potential on the surface of the rod-like polyelectrolyte with uniform charge distribution or periodically changing charge distribution on the surface is solved numerically by use of a power series method and an electronic computer. The deviation from the Linderstrøm-Lang's linear relation in the modified pH titration diagram, and the dependence of the surface electric potential on the radius of rod are discussed. Also, the radius of counterion and ionic strength, and the effect of non-uniformity of charge distribution are considered. Molecular radii were determined by comparing the calculated results with the pH titration data. The molecular radii of stereoregular poly-(methacrylic acid) and α-helical poly(glutamic acid) were found to be 5.5 Å and 15.0 Å, respectively, assuming uniform charge distribution. If nonuniform charge distribution was assumed, the molecular radii were calculated to be 5.0 Å and 14.0 Å, respectively. These values are discussed in relation to their molecular structures.     

Enantiomeric resolution by HPLC of axial chiral amides using amylose tris[(S)-1-phenylethylcarbamate]

The enantiomeric resolution of a series of N-arylamides was examined on amylose tris[(S)-1-phenylethylcarbamate] coated onto aminopropylated 7 μm silica with 500 Å diameter pores and on naked silica 5 μm particle size with 500 Å diameter pores. The enantiomeric resolution obtained for this series was excellent on both columns. The enantioselectivity of cellulose and amylose tris (3,5-dimethylphenylcarbamate) coated onto APS-Hypersil (120 Å pore size, 5 μm particle size) was also investigated for this series of compounds. Chirality 9:109–112, 1997. © 1997 Wiley-Liss, Inc.     

X-ray scattering of the non-isometric Bacillus subtilis phage phi29.

The non-isometric virus φ29 and its empty capsid have enough elements of symmetry so that their size and approximate shape can be determined by low-angle X-ray scattering. The scattering curve of the virus can be simulated by a cylinder of 390 Å diameter and 460 Å height. The protein coat has a thickness of about 30 Å. The DNA is packed tightly and regularly in the phage head.     

Production of ultrafine gold and silver particles by means of a gas evaporation and solvent trap technique

Gold (Au) and Silver (Ag) colloids were produced in ethanol by a gas evaporation technique combined with a solvent trap method. Both the clusters in the solution and those collected before entering the solution were examined by transmission electron microscopy. The size of colloids in ethanol was 30 ∼ 100Åfor Au and 50 ∼ 200Åfor Ag. In contrast, the clusters collected before the solution trap had a dimension of 10 to 60Å, which is smaller than those trapped in ethanol. This size increase was brought about by coalescent growth in the solution, which took place when the solution temperature was raised from dry ice/acetone temperature to room temperature.     

X-ray microdiffraction reveals the orientation of cellulose microfibrils and the size of cellulose crystallites in single Norway spruce tracheids

The microfibril angle (MFA) distribution and the size of cellulose crystallites in isolated double cell walls of Norway spruce (Picea abies [L.] Karst.) tracheids were determined by synchrotron X-ray microdiffraction using the reflections 200 and 004. Samples were 25 μm thick longitudinal sections of earlywood from annual rings 6–18 of several stems. The asymmetric MFA distributions extended from ?20° to 90°. The mean MFA of tangential cell walls decreased from an average of 24° into 19° from the pith to the bark. The mode of the MFA distribution was about 10° smaller than the mean MFA. The standard deviation of the MFA distribution varied between 18° and 25°. The mean MFA and the mode of the MFA distribution were larger in radial than in tangential cell walls. MFA distributions of mature wood samples exhibited a separate small peak at around 90°. The average width and length of cellulose crystallites varied between 28.9–30.9 Å and 192–284 Å, respectively. Both increased slightly as a function of annual ring number from the pith up to the 15th annual ring. An irrigation–fertilisation treatment of some of the stems resulted in longer cellulose crystallites compared to the untreated stems.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

Distribution of antibodies to the troponin complex,troponin-C and troponin-I in chicken skeletal muscle as determined by a simplified method for immuno-electron microscopy

Antisera against the troponin complex, troponin-C and troponin-I have been utilized to locate these proteins in normal, adult chicken skeletal muscle and in filaments prepared from chicken acetone dried powder. The antisera had been previously characterized by immunochemical methods and were employed to ascertain the distribution of the proteins by a simple method for immuno-electron microscopy. Glycerinated chicken breast muscle was treated with the antisera, unreacted antibody was washed from the muscle, and a goat anti-rabbit γ-globulin was added to enhance the electron density of the antigen-antibody complexes. A periodic distribution of anti-troponin-C at a mean interval of 389 Å was observed along the thin filaments in the sectioned tissue. Anti-troponin-I was deposited every 399 Å (P < 0.01). Thin filaments were prepared from acetone dried powder and reacted with the antisera. The anti-troponin-C was located every 386 Å; anti-troponin-I, every 399 Å (P < 0.01). Our technique for immuno-electron microscopy is compared with that used by others, and the significance of the findings is discussed.     

Freeze-fracture study of water-soluble,standard proteins and of detergent-solubilized forms of sarcoplasmic reticulum Ca2+-ATPase

Conventional freeze-fracture electron microscopy was used to study water-soluble proteins and different forms of Ca²⁺-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was close to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca²⁺-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particles sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca²⁺-ATPase. The results can be interpreted on the assumption that the Ca²⁺-ATPase molecule is elongated, with an overall length of about 110 Å and a width in its largest part of about 75 Å. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution.     

Cold-sensitive regulatory mutants of simian virus 40

A preparation of short synthetic myosin filaments (minifilaments) in the absence of other myosin forms is reported. Myosin minifilaments have been prepared by dialysing myosin from vertebrate striated muscle into 10 mm-citrate/Tris buffer (pH 8.0 at 4 °C) containing no other salt. These polymers of myosin are very stable and show little tendency to aggregate or dissociate in the original solvent. Sedimentation velocity, diffusion and viscosity measurements indicate that the minifilaments are composed of 16 to 18 molecules. Examination of electron micrographs reveals that the bare central region of minifilaments extends over 1600 to 1800 Å and the entire particles are about 3000 Å long with a diameter of 80 Å across the smooth region. They have the appearance of short bipolar filaments (Huxley, 1963). In solution the minifilaments are homogeneous in terms of size distribution and exhibit normal MgATPase and CaATPase activities. When examined in the ultracentrifuge, the minifilaments sediment in the form of a hypersharp peak (or bar) with a sedimentation coefficient independent of rotor speed. The minifilaments can be dissociated by ATP, hardly by MgATP; whereas KCl (between 0.04 and 0.2 m) induces further polymerization. It is suggested that the minifilaments are an intermediate in the assembly of myosin filaments.     

Distance distributions in native and random-coil troponin I from frequency-domain measurements of fluorescence energy transfer

We used time-dependent fluorescence energy transfer to determine the distribution of donor-to-acceptor distances in native and denatured troponin I(TnI). The single tryptophan residue (Trp 158) of TnI served as the donor (D), and the acceptor (A) was a labeled cysteine residue (Cys 133). The time-dependent intensity decays of the donor were measured by the frequency-domain method from 10 to 320 MHz. The frequency response of the donor emission, in the absence and presence of acceptor, was used to recover the distribution of D to A distances, using an algorithm that accounts for the intrinsic multiexponential decay of the donor. In the native state the D–A distribution is characterized by an average distance of 23 Å and a half-width of 12 Å. Denaturation results in a modest increase in the average distance to 27 Å, and a dramatic increase in half-width to 47 Å. We believe the ability to recover distance distributions will have numerous applications in the characterization of biological macromolecules.     

Structure of human serum lipoproteins in solution. II. Small-angle x-ray scattering study of HDL and LDL.

Two human serum lipoprotein particles, HDL₃ and LDL, were studied in solution in solvents of variable density (NaBr in water) by small-angle X-ray scattering using a position-sensitive proportional counter. The data were analysed using the theoretical approach outlined in the accompanying paper (Luzzati et al., 1976). The structures of the particles were found to be independent of the salt concentration of the solution (i.e. the particles are impenetrable to NaBr). Density heterogeneities are negligible and size and shape heterogeneities appear to be small.The particle structures could be quantitatively described in terms of a set of parameters and of a few one-dimensional functions. The parameters are the volume, radius of gyration and surface area of the shape functions; the second moment and square average of the electron density contrast at buoyancy; the electron density level, volume, radius of gyration and surface area of the hydrocarbon and polar regions. The one-dimensional functions are: the distribution of chords, the spherical average of the shape function and of the electron density at buoyancy, and the fraction of each spherical shell occupied by the hydrocarbon and polar regions. These parameters and functions are internally consistent and agree with the chemical data confirming the assumptions made in their derivation.The results are compatible with the shape of the particle being compact and quasi-spherical although with deeply convoluted surfaces. They also indicate that the outer layers of the particles are occupied by the proteins and the polar groups of phospholipids and free cholesterol, and the cores by neutral lipids. The maximum diameters of the particles are 130Å and 280Å for HDL₃ and LDL, respectively, while the hydrocarbon cores have diameters of 80Å and 230Å, respectively. The solvent is considered to penetrate to 25Å from the center of the HDL₃ particle with a minimum solvation at a radius of 45Å. In the case of LDL, the solvent penetrates to 55Å from the center of the particle. The lipids in the cores of the particles, particularly the cholesterol esters, appear to display a micelle-like organization with the steroid nuclei segregated in regions distinct from those occupied by the hydrocarbon chains.Although the data are consistent with several aspects of previously proposed models, they indicate that the structures of the HDL₃ and LDL particles are more complex than previously believed.     

Lamellar dispersion and phase separation of chloroplast membrane lipids by negative staining electron microscopy

Aqueous dispersions of lipids isolated from spinach chloroplast membranes were studied by electron microscopy after negative staining with phosphotungstic acid. Influence of low temperature (5°C for 24 h) was also investigated. It was observed that when contacted with water, these lipids, as such, formed multilamellar structures. Upon sonication, these multilamellar structures gave rise to a clear suspension of unilamellar vesicles varying in size (diameter) between 250 and 750 Å. When samples of sonicated unilamellar vesicles were stored at 5°C for 24 h or more, they revealed a variety of lipid aggregates including liposomes, cylindrical rods (about 100 Å wide and up to 3600 Å long), and spherical micellar structures (100–200 Å in diameter)—thus indicating phase separation of lipids.     

Changes in the x-ray solution scattering of aspartate transcarbamylase following the allosteric transition.

Aspartate transcarbamylase (Escherichia coli) has been studied by X-ray solution scattering in the s range 0.002 to 0.06 Å^?1. The spectra display sharp maxima and minima whose positions and amplitudes show considerable changes upon ligation with the transition state analogue N-(phosphonacetyl)-l-aspartate. The magnitude of the change in diffraction pattern is so large that X-ray solution scattering should be a useful technique for studying the proportions of different quaternary forms in solutions of this enzyme. In particular, the kinetics of the allosteric transition appear to be within the reach of X-ray diffraction experiments.Some structural parameters of the allosteric transition were obtained from the diffraction patterns. The radius of gyration of the native enzyme is 45.9 ± 0.5 Å, and after ligation it increases to 48.4 ± 1.0 Å. At the same time, the peak of the pair distribution function is shifted from 58 Å to 63 Å. These changes indicate that the molecule swells after the allosteric transition to the R form. However, the maximum distance (from the pair distribution function) does not increase after ligation, and may even decrease slightly. Some probable subunit movements during allosteric activation are discussed.     

Crystallization and preliminary X-ray diffraction analysis of bovine seminal plasma PDC-109, a protein composed of two fibronectin type II domains

PDC-109 is a 13 kDa glycoprotein and the major phosphorylcholine- and heparin-binding protein of bull seminal plasma. It is built by an acidic 23-residue N-terminal sequence followed by a tandem of fibronectin type II domains. Full-length PDC-109 was crystallized in complex with o-phosphorylcholine by vapor diffusion in sitting drops. Crystals grew to maximal size of 0.5 × 0.3 × 0.2 mm³, diffract x-rays beyond 2.6 Å resolution, and belong to space group P321 with unit cell dimensions a = b = 93.6 Å, c = 52.7 Å. Proteins 28:454–456, 1997. © 1997 Wiley-Liss, Inc.     

Biogeography of the smooth snake (Coronella austriaca): origin and conservation of the northernmost population

Understanding historical range expansions and population demography can be crucial for the conservation and management of endangered species. In doing so, valuable information can be obtained regarding, for example, the identification of isolated populations, associations to particular habitats and distribution range shifts. As poikilotherms, snakes are vulnerable to environmental changes that can greatly shape their distribution ranges. Here we used mitochondrial data to elucidate the origin of the smooth snake population in Åland island, which is the northernmost location where the species is found. In Åland, we used mitochondrial and microsatellite data to fine‐map its spatial genetic structure, infer its demographic dynamics and determine its effective population size. We found three independent lineages, which expanded north from Iberian, the Balkans and Caucasus regions. The central lineage originating in the Balkans was the only one that reached Scandinavia. The Åland population belongs to this lineage and potentially colonized the island from the west via Sweden. This population appeared to be critically small and fragmented into two genetically isolated subpopulations. We discuss our results in light of previous findings regarding colonization routes in Europe and Scandinavia. Moreover, we discuss the origin and current genetic status of the Åland population relative to other co‐occurring snakes and suggest conservation measures based on our findings. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 426–435.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.