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B L Trus

Publications and source records attributed to B L Trus.

At least 73 records · Page 4Linked to original sources
PubMed1985

The cytoplasmic matrix: its volume and surface area and the diffusion of molecules through it.

In this work we look into the problem of why proteins, unlike small molecules, diffuse in the cytoplasm much more slowly than in aqueous solutions. In order to examine whether the cytoplasmic matrix could, by simple obstruction, retard protein diffusion to such an extent, we developed a method to measure semiquantitatively the fractional volume occupied by the cytoplasmic matrix (which includes the microfilaments, intermediate filaments, microtubules, and the microtrabeculae of the cytoplasmic matrix). This method yielded values in the range of only 16-21%. Thus, a more elaborate model is suggested in which the diffusing proteins bind and dissociate constantly from the surfaces in the cytoplasmic matrix. From this model, the diffusion coefficients and the measured values of the fractional volumes, we calculated the corresponding binding constants. These values indicate that most of the diffusing proteins are bound to the matrix at any given time, in spite of the possibility that they may bind and dissociate very rapidly. In addition, from our measurements, we estimate the surface area of structures within the cytoplasmic matrix to be in the range of 69,000-91,000 micron 2 per cell.

Animals↗
PubMed1985

Presence of two distinct regions in the coiled-coil structure of the streptococcal Pep M5 protein: relationship to mammalian coiled-coil proteins and implications to its biological properties.

The complete amino acid sequence of Pep M5, a biologically active 197-residue fragment comprising nearly half of the group A streptococcal M5 protein, has structural features characteristic of an alpha-helical coiled-coil protein. Fourier analyses of the nonpolar residues show strong periodicities based on repeats of 7 residues (7/2 and 7/3). Except for the nonhelical NH2-terminal 12-residue segment, the 7-residue periodicity in the distribution of nonpolar residues extends through the remainder of the Pep M5 molecule, with some discontinuities and irregularities. The molecule contains two distinct regions that differ in the pattern of distribution of the nonpolar and charged residues. The 7-residue pattern "a, b, c, d, e, f, g" in region 13-121 is atypical in that position "a" is predominantly occupied by asparagine, rather than nonpolar residues. On the other hand, the periodicity in region 122-196 is more typical of that found in other coiled-coil proteins, such as the myosin rod region, keratin, desmin, and vimentin, rather than tropomyosin. Although the periodicity in nonpolar residues is not highly regular, the predominance of basic and acidic residues in the inner "e" and "g" positions, respectively, suggests that ionic interactions between chains may contribute significantly to the stability of the coiled-coil. The distribution of charged residues in the outer positions within the two regions of the molecule is also distinct. The NH2-terminal region carries a significantly higher net negative charge than the COOH-terminal region, suggesting that the former region may play an important role in some of the biological functions of the Pep M5 molecule.

Amino Acid Sequence↗
PubMed1985

Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.

Capsid↗
PubMed1984

Diffraction patterns from stained and unstained helices: consistency or contradiction?

Addressing the issue of how best to integrate negative staining electron microscopy with X-ray fiber diffraction of macromolecular helices, we investigated staining properties of helical models using computational simulations. We compared diffraction patterns of stained and unstained representations of F-actin, myosin S1-decorated actin, and microtubules, to test the reliability of selecting well preserved specimens as those whose optical diffraction patterns most closely match the X-ray patterns of hydrated fibers. We conclude that if the stain layer is considerably thicker than the outer diameter of the specimen, this criterion is likely to be reliable. However, specimens in such thick stain layers may be resolution-limited by dynamic scattering effects such as beam-broadening. With relatively thin staining (the more practically relevant situation) "edge effects" in the stain distribution result in differences between diffraction patterns of stained and unstained specimens. Diffraction pattern changes due to molecular distortions such as shrinkage or flattening were similarly modeled. Since differences between diffraction patterns of stained and unstained helices may be due to either "edge effects" or molecular distortions, it does not appear possible a priori to distinguish between these effects. Comparison of experimental data for the (2.3 nm)-1 layer-line of tobacco mosaic virus (as model system) reveals major differences between the X-ray diffraction pattern of hydrated sols and the Fourier transform of HREM images of negatively stained specimens.

Microscopy, Electron↗
PubMed1984

The complete cDNA and deduced amino acid sequence of a type II mouse epidermal keratin of 60,000 Da: analysis of sequence differences between type I and type II keratins.

We present the complete nucleotide and deduced amino acid sequences of a mouse epidermal keratin subunit of 60,000 Da. The keratin possesses a central alpha-helical domain of four tracts (termed 1A, 1B, 2A, and 2B) that can form coiled-coils, interspersed by short linker sequences, and has non-alpha-helical terminal domains. This pattern of secondary structure is emerging as common to all intermediate filament subunits. The alpha-helical sequences conform to the type II class of keratins. Accordingly, this is the first type II keratin for which complete sequence information is available, and thus it facilitates elucidation of the fundamental distinctions between type I and type II keratins. It has been observed that type I keratins are acidic and type II keratins are neutral--basic in charge. We suggest that the basis for this empirical correlation between type and charge resides in the respective net charges of the 1A and 2B tracts. Calculations on interchain interactions between charged residues in the alpha-helical domains indicate that this keratin prefers to participate in dimers according to an in-register parallel arrangement. The terminal domains of this keratin possess characteristic glycine-rich sequences, and the carboxyl-terminal domain is highly homologous to that of a human epidermal keratin of 56,000 Da. According to the hypothesis that end-domains are located on the periphery of keratin filaments, we conclude that the corresponding mouse and human keratins are closely related, both structurally and functionally.

Amino Acid Sequence↗
PubMed1984

Radial distributions of density within macromolecular complexes determined from dark-field electron micrographs.

A procedure has been developed for direct determination of radial distributions of density in filamentous and spheroidal particles by analyzing dark-field scanning transmission electron micrographs of unstained freeze-dried specimens. Unlike electron microscopic methods based on staining or shadowing with heavy atoms, this approach can be used to probe the internal structure of macromolecular complexes. As an experimental proving ground, we have applied the procedure to tobacco mosaic virus (TMV) and to RNA-free helical polymers of TMV coat protein. Both structures are found to project outermost diameters of 17.6 +/- 0.4 nm, to have empty axial holes approximately equal to 3.5 nm in diameter, and to have density peaks at radii of 2.5 +/- 0.5 and 6.7 +/- 0.3 nm. Thus visualized, the only significant difference between them is the presence in the virion of an additional density peak at 4.1 +/- 0.5 nm contributed by its internalized RNA molecule. We have also used the procedure to monitor the structural expression of radiation damage in the low electron dose regime prior to the onset of significant mass loss. Changes in the radial density profiles are detected at average doses as low as approximately equal to 400 electrons per nm2: the trend is for the internal structure of these particles to fuse toward a state of uniform density, although the values of their outermost diameters remain unaffected.

Densitometry↗
PubMed1983

The distribution of mass in heteropolymer intermediate filaments assembled in vitro. Stem analysis of vimentin/desmin and bovine epidermal keratin.

We have studied the distribution of mass in several types of intermediate filaments (IF) assembled in vitro, by analyzing scanning transmission electron micrographs (STEM) of unstained specimens imaged in dark-field mode. Bovine epidermal keratin IF, which are obligate heteropolymers, were thus characterized and compared with facultative heteropolymers of vimentin and desmin and with earlier observations of homopolymer vimentin IF. The major components of each type of IF have linear densities of 37 kilodaltons/nm. Minor polymorphic variants are also present in each case, with linear densities of approximately 25 and 48 kilodaltons/nm, respectively. In view of the known subunit masses, these results are consistent with the proposition that at least three IF types (bovine epidermal keratin, vimentin, and desmin) are structurally homologous. For each type of IF studied, measurements taken near the ends of long IF or from short IF tend to have lower densities, characteristic of the least dense polymorphic variant. This form may represent a precursor "minimal core" structure in the sense of a minimal aggregate of protofilaments sufficiently stable to permit elongation. In the course of in vitro assembly, this form would be maturable to the normal IF structure at 37 kilodaltons/nm by the accretion of further protein. We also find the projected widths of these IF as measured from the STEM images to be significantly greater than the corresponding diameters determined in previous studies by conventional electron microscopy. Whereas the conventional techniques have routinely yielded diameters of 7-11 nm for IF contrasted by heavy metal staining, the STEM images of unstained IF provide estimates of 15 +/- 1 nm for the outer diameter of the major density class for each IF type studied and average values of 13.8 and 16.1 nm for the less dense and more dense variants, respectively.

Animals↗
PubMed1983

Complete amino acid sequence of a mouse epidermal keratin subunit and implications for the structure of intermediate filaments.

We have determined the complete primary structure of an intermediate filament subunit, the 59,000 molecular weight subunit of mouse epidermal keratin, from the nucleotide sequence of cDNA clones. The central portion of the sequence forms extended tracts of a coiled-coil alpha-helical conformation. This is flanked at both termini by similar non-alpha-helical sequences that are extremely rich in glycine residues, frequently configured in tandem peptide repeats. Limited chymotryptic digestion of keratin filaments containing this protein suggests a structural organization whereby the terminal glycine-rich sequences protrude from a conserved core structure into which the coiled-coil alpha-helical segments are packed.

Amino Acid Sequence↗
PubMed1983

Quantitative double-label radiography of two-dimensional protein gels using color negative film and computer analysis.

We have devised a method of data collection and computer analysis which allows utilization of the resolving power of two-dimensional gel electrophoresis of proteins, in conjunction with the versatility of using two different radionuclides simultaneously. Cultures of Escherichia coli growing with exponential growth rate constants (mu) of 0.32 and 1.43 were labeled with [3H]leucine and [14C]leucine, respectively; these samples were mixed, and cell protein was separated on a two-dimensional gel. Spacial and quantitative data for both radionuclides were recorded on color negative film by radiographic exposure. Data for 14C alone were then collected photographically from the red-light-sensitive layer of the film using a red filter, while data for 3H and spillover of 14C were collected photographically from the blue-light-sensitive layer using a blue filter. These two data sets were analyzed by CINT, a computer program for analysis of two-dimensional gels, and quantitative data for 3H were calculated after determination of spillover of 14C in a manner analogous to quantification of 3H and 14C by liquid scintillation counting. Quantitative data from over 1000 protein spots representing from 0.002% to 10% of the total 3H or 14C, respectively, are available in a matter of hours. We have used this method to analyze the effect of growth rate and medium composition on the relative levels of individual proteins in a pathogenic strain of E. coli which contains group 111 O-antigen. As expected, the relative levels of aminoacyl-tRNA synthetases, protein chain elongation factors, ribosomal proteins, and the alpha-subunit of RNA polymerase are all increased with increased growth rate; the magnitude of these changes agreed with previous data derived using other strains of E. coli. Alterations in the levels of other proteins identified on the two-dimensional gels could be interpreted in terms of changes in medium composition. When compared to manual data collection by excising radiolabeled proteins and quantifying 3H and 14C in a liquid scintillation counter following combustion to H2O and CO2, respectively, this new method of data collection and computer analysis increases the resolution of data collection and decreases the time involved from days to hours.

Bacterial Proteins↗
PubMed1983

Molecular architecture of bacteriophage T7 capsid.

To determine the capsid structure of bacteriophage T7, we have investigated polycapsids, tubular capsid-related structures isolated from lysates of the T7 mutant am16. Biochemical analysis shows polycapsids to be composed of gp10, the major structural protein of the wild-type capsid. The conformational state of gp10 in polycapsids is indistinguishable from that in the mature virus capsid by the criteria of surface charge, buoyant density, and insensitivity to proteolysis by trypsin. Optical diffraction of electron micrographs of negatively stained polycapsids reveals a hexagonal surface lattice of periodicity 12.6 +/- 0.2 nm and is used to analyze the distribution of cylindrical foldings of this lattice into polycapsids (polymorphic variation). These foldings are found to be related to that of the capsid proper through the intrinsic curvature of gp10, each folding having a set of lattice lines whose radius of curvature is close to 29 nm. The fine structure of this surface lattice has been elucidated by digital image processing of electron micrographs. The capsomer is shown unequivocally to be a hexamer of characteristic morphology. By collating these results with earlier observations, we conclude that the structure of the normal T7 capsid is an orthodox icosahedron of triangulation class T = 7, composed of 60 hexamers and 12 pentamers.

Capsid↗
PubMed1983

The effects of thyroid hormone on in vitro phosphorylation, acetylation, and ADP ribosylation of rat liver nuclear proteins.

The effect of thyroid hormone on acetylation, phosphorylation and ADP ribosylation of rat liver nucleoproteins was studied by incubating intact nuclei with labeled precursors. Acetylation, which occurred in histones and low molecular weight proteins (less than 30,000), was depressed in nuclei from thyroidectomized animals. The administration of L-3,5,3'-triiodothyronine (T3) increased acetate incorporation to 50% over control levels. Incorporation of labeled phosphate from ATP into most proteins was decreased in nuclei from thyroidectomized animals and increased by the administration of T3. The greatest increase produced by T3 (to 140% of control values) was seen in proteins of molecular weight greater than 68,000. Nuclei from thyroidectomized animals incorporated less ADP ribose in most proteins. Both high molecular weight proteins (greater than 68,000) and low molecular weight proteins (less than 30,000) showed a further decrease in ADP ribose incorporation in nuclei from thyroidectomized rats given T3. However, a few proteins of the middle molecular weight class showed increased ADP ribose incorporation subsequent to the injection of T3. It is suggested that a generalized increase in protein synthetic rates previously noted to be caused by T3 is accompanied by increased acetylation and phosphorylation of histones and other proteins. These changes could accelerate transcription of already active genes.

Acetylation↗
PubMed1983

Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments.

We have used scanning transmission electron microscopy to elucidate the question of how intermediate filament (IF) subunits of widely differing mass can all form morphologically similar IF. From scanning transmission electron micrographs, the distributions of mass were determined for three types of epidermal keratin IF reassembled in vitro from mixtures of subunits with substantially different masses, viz., "light" and "heavy" human keratins with [Mr] = 50,000 and 56,000, respectively, and mouse keratins of [Mr] = 63,000. Their principal assembly products were found to average 22, 25, and 29 kdalton/nm, respectively. These densities, which correspond to immature "minimal form" IF (Steven, A. C., J. Wall, J. Hainfeld, and P. M. Steinert, 1982, Proc. Natl. Acad. Sci. USA., 79:3101-3105), are directly proportional to the average subunit masses. The human keratin IF (but not those of mouse) also contained minor amounts (15-20%) of more massive polymers averaging 33 and 35 kdalton/nm, respectively, which probably represent mature IF. Taken together with earlier results on IF of other subclasses, these results indicate that the average linear density of IF scales according to the average mass of their constituent subunits, both for "minimal form" and for mature IF. As underlying mechanism for this homology, we propose that the fundamental building-blocks of all these IF contain a common structural element whose packing within the various IF is likewise conserved and which specifies the overall structure. The variable amounts of mass in the nonconserved moieties account for the observed proportionality. This scheme fits with amino acid sequence data for several IF subunits that have revealed, as a likely candidate for the common element, an essentially conserved alpha-helical domain, contrasting with the highly variable sequences of their non-alpha-helical terminal domains.

Animals↗
PubMed1981

A new model for packing of type-I collagen molecules in the native fibril.

A specific fibril model is presented consisting of bundles of five-stranded microfibrils, which are usually disordered (except axially) but under lateral compression become ordered. The features are as follows (where D = 234 residues or 67 nm): (1) D-staggered collagen molecules 4.5 D long in the helical microfibril have a left-handed supercoil with a pitch of 400-700 residues, but microfibrils need not have helical symmetry. (2) straight-tilted 0.5-D overlap regions on a near-hexagonal lattice contribute the discrete x-ray diffraction reflections arising from lateral order, while the gap regions remain disordered. (3) The overlap regions are equivalent, but are crystallographically distinguished by systematic displacements from the near-hexagonal lattice. (4) The unit cell is the same as in a recently proposed three-dimensional crystal model, and calculated intensities in the equatorial region of the x-ray diffraction pattern agree with observed values.

Animals↗
PubMed1981

Two-dimensional gel analysis of rat liver nuclear proteins after thyroidectomy and thyroid hormone treatment.

The composition of nucleoproteins in normal rat liver, in livers from thyroidectomized animals, and in thyroidectomized animals treated with 3,3',5-triiodo-L-thyronine was examined by two-dimensional gel electrophoresis. A computer program was designed to measure the relative concentrations of each protein. Approximately 500 protein subunits could be distinguished. Of these, 102 either disappeared or were markedly decreased after thyroidectomy. Shortly after administration of the thyronine (6 hr), 13 proteins reappeared and, by 24 hr, 67 additional proteins had reappeared. These changes in protein concentrations are both the greatest in number and have the largest reported as an effect of thyroid hormone. Twenty-four hr after triiodothyronine administration, some 22 proteins seen in normal liver could not be found. Two new proteins appeared in thyroidectomized rat liver nuclei. We conclude that a major and rapid effect of thyroid hormone is exerted on hepatic nucleoproteins.

Animals↗
PubMed1980

Compressed microfibril models of the native collagen fibril.

A three-dimensional crystal model for packing of collagen molecules (type I) in the native fibril has recently been proposed by Hulmes and Miller. It provides a straightforward explanation of the major features of the X-ray diffraction pattern, and is consistent with measurements of fibril density. However, there is independent evidence for a well defined microfibrillar substructure, which is absent from their model. This evidence, which is derived from electron microscopy and studies of in vitro assembly, the pattern of covalent crosslinks and sequence analysis, is convincing. Therefore, we have searched for a means to reconcile this conflict. We now propose two models which contain five-stranded microfibrils compressed to place molecules (in cross-section) on a pseudohexagonal lattice. The unit cells are equivalent or related to the cell proposed by Hulmes and Miller. In the simplest case, molecules, and thus microfibrils, are straight-tilted. However, it is not ruled out that molecules are supercoiled and microfibrils are straight. Noncrystallographic considerations favour supercoiling.

Collagen↗
PubMed1977

Tuna cytochrome c at 2.0 A resolution. III. Coordinate optimization and comparison of structures.

Optimum coordinate sets have been obtained for ferrocytochrome c and the two symmetry-independent molecules of ferricytochrome c from tuna at 2.0 A resolution by making the best fit of models with standard bond lengths and angles to the experimental electron density maps (1977) J. Biol. Chem. 252, 759-785, as a preliminary to full refinement with 1.5 A data. Both the Diamond model-building programs and locally developed minicomputer routines were tried, with the latter preferred for economy and ease of operation, although both gave satisfactory results. Atomic coordinates are available on microfiche or from the Brookhaven Protein Data Bank. Using the two ferricytochrome molecules as a control, no differences between oxidized and reduced cytochrome molecules can be seen that are outside the probable limits of accuracy of the 2.0 A analysis. Rotation and subtractive difference map comparisons also show no conformation changes. If believable differences do appear in the course of the 1.5 A refinement now underway, these should be no more than minor breathing of main chain or adjustment of side chains.

Amino Acid Sequence↗