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Biomedical subjects

V Ramakrishnan

Publications and source records attributed to V Ramakrishnan.

At least 181 records · Page 10Linked to original sources
PubMed1990

Interaction of HMG14 with chromatin.

Neutron scattering has been used to study the interaction of HMG14 with chromatin. Chromatin depleted of H1/H5 was reconstituted separately with histones H1 and H5, and complexed with HMG14. We have also studied the conformation of complexes formed by the binding of HMG14 to nucleosome dimers without linker DNA. Our data on the binding of HMG14 to linkerless nucleosome dimers argue against a significant change in the exit and entry angles of nucleosomal core DNA. Data on the condensation of chromatin into a higher-order structure suggest that there is no dramatic difference between the roles of H1 and H5 in their influence on HMG14 complex formation. However, there is a decrease of about 25% in the mass per unit length of chromatin fibers on HMG14 binding, which is not accompanied by a change in the fiber repeat distance. This is evidence that there are fewer nucleosomes per repeat in HMG14 containing chromatin fibers than in normal chromatin. Alteration of chromatin structure in this manner may be part of the role of HMG14 in actively transcribed chromatin.

Animals↗
PubMed1990

Characterization of an extremely large, ligand-induced conformational change in plasminogen.

Native human plasminogen has a radius of gyration of 39 angstroms. Upon occupation of a weak lysine binding site, the radius of gyration increases to 56 angstroms, an extremely large ligand-induced conformational change. There are no intermediate conformational states between the closed and open form. The conformational chang is not accompanied by a change in secondary structure, hence the closed conformation is formed by interaction between domains that is abolished upon conversion to the open form. This reversible change in conformation, in which the shape of the protein changes from that best described by a prolate ellipsoid to a flexible structure best described by a Debye random coil, is physiologically relevant because a weak lysine binding site regulates the activation of plasminogen.

Aminocaproic Acid↗
PubMed1990

Crystallization of the globular domain of histone H5.

The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed in Escherichia coli a gene that codes for the globular domain of H5. The protein produced in E. coli is functional in nucleosome binding assays. We have obtained crystals of the protein that diffract to beyond 2.5 A (1 A = 0.1 nm) resolution. The crystals are orthorhombic with unit cell dimensions of a = 80.1 A, b = 67.5 A and c = 38.0 A.

Crystallization↗
PubMed1990

Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers.

Several groups have demonstrated that radioiodinated tissue-type plasminogen activator (t-PA) binds to saturable sites on human umbilical vein endothelial cells (HUVECs) in culture (Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719; Beebe, D. P. (1987) Thromb. Res. 46, 241-254; Barnathan, E. S., Kuo, A., van der Keyl, H., McCrae, K. R., Larsen, G. L., and Cines, D. B. (1988) J. Biol. Chem. 263, 7792-7799). Here we report that most of the specific binding of 125I-t-PA to our HUVEC cultures is accounted for by binding to (i) plasminogen activator inhibitor type 1 (PAI-1), a t-PA inhibitor produced in abundance by HUVECs; and (ii) specific binding sites present on the plastic culture surface. The contribution of the sites on plastic can be eliminated by taking several precautions. Then, most or all of the specifically bound 125I-t-PA is present in a sodium dodecyl sulfate-stable 110-kDa 125I-t-PA.PAI-1 complex. Interestingly, a radioiodinated mutant form of t-PA, S478A, which is catalytically inactive and therefore unable to form the covalent complex with PAI-1, still binds to HUVECs. In fact, this ligand binds to HUVECs in 10-30-fold greater amounts than does wild-type 125I-t-PA (resulting in greater than 1 x 10(7) S478A 125I-t-PA molecules bound/cell at 12 nM ligand concentration). In contrast, diisopropyl fluorophosphate-treated t-PA binds to HUVECs in much smaller amounts than does wild-type t-PA. Several findings suggest that PAI-1 is a major binding site for S478A t-PA. The vast amount of binding observed with S478A t-PA, compared with wild-type t-PA, may be accounted for by an observed large scale release of wild-type 125I-t-PA.PAI-1 complexes from the solid phase (cells or extracellular matrix) into the culture medium. Immunoprecipitation experiments demonstrate that, in contrast to wild-type t-PA, S478A t-PA does not extract [35S]methionine-PAI antigen from metabolically labeled extracellular matrix. It is proposed that t-PA releases PAI-1 from the solid phase when it forms the irreversible covalent complex with the inhibitor, a process that does not occur with the catalytically inactive mutant form of t-PA.

Cells, Cultured↗
PubMed1988

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin.

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.

Animals↗
PubMed1987

A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli.

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data. The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.

Bacterial Proteins↗
PubMed1987

Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy.

Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and seven nucleosomes per 11 nm. There is no evidence of a transition from a 10-nm to a 30-nm fiber. Fiber diameter is correlated with mass per unit length, showing that both increase during condensation. We also find that there is no essential difference between the mass per unit length measured by scanning transmission electron microscopy and neutron scattering in solution, showing that the ordered regions seen in micrographs are representative of chromatin in solution.

Animals↗
PubMed1986

A role for proteins S3 and S14 in the 30 S ribosomal subunit.

Small ribosomal subunits prepared by the method of Kirillov et al. (Kirillov, S. V., Makhno, V. I., Peshin, N. N., and Semenkov, Yu. P. (1986) Nucleic Acids Res. 5, 4305-4315) are active but fail to reconstitute. The inability to reconstitute is due to a deficiency in proteins S3 and S14. Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product. Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.

Escherichia coli↗
PubMed1986

Distribution of protein and RNA in the 30S ribosomal subunit.

In Escherichia coli, the small ribosomal subunit has a sedimentation coefficient of 30S, and consists of a 16S RNA molecule of 1541 nucleotides complexed with 21 proteins. Over the last few years, a controversy has emerged regarding the spatial distribution of RNA and protein in the 30S subunit. Contrast variation with neutron scattering was used to suggest that the RNA was located in a central core of the subunit and the proteins mainly in the periphery, with virtually no separation between the centers of mass of protein and RNA. However, these findings are incompatible with the results of efforts to locate individual ribosomal proteins by immune electron microscopy and triangulation with interprotein distance measurements. The conflict between these two views is resolved in this report of small-angle neutron scattering measurements on 30S subunits with and without protein S1, and on subunits reconstituted from deuterated 16S RNA and unlabeled proteins. The results show that (i) the proteins and RNA are intermingled, with neither component dominating at the core or the periphery, and (ii) the spatial distribution of protein and RNA is asymmetrical, with a separation between their centers of mass of about 25 angstroms.

Escherichia coli↗
PubMed1984

Structure of the capsid of Kilham rat virus from small-angle neutron scattering.

The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 X 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H2O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, we propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H2O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H2O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H2O. We propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions.

Amino Acids↗
PubMed1984

The shape of ribulose bisphosphate carboxylase/oxygenase in solution as inferred from small angle neutron scattering.

Small angle neutron scattering of both activated and deactivated hexadecameric ribulose bisphosphate carboxylase/oxygenase from spinach revealed that its structure in solution very closely resembles that determined for the deactivated crystalline enzyme from tobacco (Baker, T.S., Eisenberg, D., and Eiserling, F. (1977) Science 196, 293-295). Scattering from both forms of the enzyme in H2O most closely fits that expected from a hollow sphere with an outer radius of 56.4 A and inner radius of 14.3 A. In D2O the enzyme's scattering profiles more closely resembled those of a hollow cylinder with an axial ratio of approximately 1.06 and outer and inner radii of 46.7 and 12.9 A, respectively. Superpositioning of model structures showed that both models determined by neutron scattering correspond quite well to that inferred from x-ray diffraction and electron microscopy of the tobacco enzyme. Sedimentation velocity studies confirmed that no major conformational change occurs upon activation of the spinch carboxylase, both forms exhibiting a sedimentation coefficient, 8(20,w), of 17.8 S. Neutron scattering by activated and deactivated carboxylase from Rhodospirillum rubrum, a dimeric enzyme, best matched that predicted for a solid prolate ellipsoid or cylinder. The estimated dimensions of the model structures correspond strikingly well to those predicted for adjacent pairs of large subunits in the hexadecameric tobacco enzyme.

Molecular Weight↗
PubMed1983

Neutron scattering studies of nucleosome structure at low ionic strength.

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.

Animals↗
PubMed1980

Arterial embolization: problems of source, multiplicity, recurrence, and delayed treatment.

The clinical course of 225 patients who had experienced 309 arterial embolic episodes was reviewed with special reference to the problems of the identification of the source of embolization, multiplicity of involvement, significance of recurrence, and the effects of some therapeutic means, including anticoagulation, on the early and late results. The most common source of embolization was cardiac (in 85% of the cases). Persistent search, at times by complex diagnostic methods, uncovered the origin in all but 11% of the cases. Within a range of 8 hours to 7 days the effect of delayed treatment had a linear relationship to the severity of ischemic changes and the deterioration of favorable results. Angiography was helpful and often essential in the diagnosis of visceral embolism and in differentiating local thrombosis from embolism in the periphery. Recurrent embolization was common (in 28% of the cases), was often multiple, and had a grave prognosis. Permanent anticoagulant therapy, preferably with but even without the removal of the source of embolization, reduced the rate of recurrence and, in general, reduced early and late morbidity and mortality rates.

Adult↗