Nucleotide sequence of the 3terminal region of belladonna mottle virus (renamed Physalis mottle virus) RNA and analysis of the evolutionary relationships among tymoviral coat proteins

The 3' terminal 1255 nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3' terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addiition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

The crystal structure of the dipotassium salt of uridine 5'-diphosphate

C9H12N2Ot2P22-. 2K + .3H20 is orthorhombic, P2~2~2p with a = 18.977 (5), b - 22.597 (6), c = 8.995 (2) A, Z = 8. The structure was refined to R = 0.059 for 2587 observed reflexions. The two molecules of the asymmetric unit have very similar conformations with a 2'- endo sugar pucker and a folded pyrophosphate chain. They form a dimer, coordinated by the K + ions but without direct bridging between the base and the pyrophosphate within each individual molecule. One uracil base has the keto-enol and the other the diketo form. The extended structure shows alternating hydrophobic and hydrophilic regions.

Interaction Energy Based Protein Structure Networks

The three dimensional structure of a protein is formed and maintained by the noncovalent interactions among the amino acid residues of the polypeptide chain These interactions can be represented collectively in the form of a network So far such networks have been investigated by considering the connections based on distances between the amino acid residues Here we present a method of constructing the structure network based on interaction energies among the amino acid residues in the protein We have investigated the properties of such protein energy based networks (PENs) and have shown correlations to protein structural features such as the clusters of residues involved in stability formation of secondary and super secondary structural units Further we demonstrate that the analysis of PENs in terms of parameters such as hubs and shortest paths can provide a variety of biologically important information such as the residues crucial for stabilizing the folded units and the paths of communication between distal residues in the protein Finally the energy regimes for different levels of stabilization in the protein structure have clearly emerged from the PEN analysis

Temperature-Sensitive Src-Transformed Mdck Cells nn 3d Cultures as a Model of Malignant Transformation of Epithelial Cells

The equilibrium between cell proliferation, differentiation, and apoptosis is crucial for maintaining homeostasis in epithelial tissues. In order for the epithelium to function properly, individual cells must gain normal structural and functional polarity. The junctional proteins have an important role both in binding the cells together and in taking part in cell signaling. Cadherins form adherens junctions. Cadherins initiate the polarization process by first recognizing and binding the neighboring cells together, and then guiding the formation of tight junctions. Tight junctions form a barrier in dividing the plasma membranes to apical and basolateral membrane domains. In glandular tissues, single layered and polarized epithelium is folded into tubes or spheres, in which the basal side of the epithelial layer faces the outer basal membrane, and the apical side the lumen. In carcinogenesis, the differentiated architecture of an epithelial layer is disrupted. Filling of the luminal space is a hallmark of early epithelial tumors in tubular and glandular structures. In order for the transformed tumor cells to populate the lumen, enhanced proliferation as well as inhibition of apoptosis is required. Most advances in cancer biology have been achieved by using two-dimensional (2D) cell culture models, in which the cells are cultured on flat surfaces as monolayers. However, the 2D cultures are limited in their capacity to recapitulate the structural and functional features of tubular structures and to represent cell growth and differentiation in vivo. The development of three-dimensional (3D) cell culture methods enables the cells to grow and to be studied in a more natural environment. Despite the wide use of 2D cell culture models and the development of novel 3D culture methods, it is not clear how the change of the dimensionality of culture conditions alters the polarization and transformation process and the molecular mechanisms behind them. Src is a well-known oncogene. It is found in focal and adherens junctions of cultured cells. Active src disrupts cell-cell junctions and interferes with cell-matrix binding. It promotes cell motility and survival. Src transformation in 2D disrupts adherens junctions and the fibroblastic phenotype of the cells. In 3D, the adherens junctions are weakened, and in glandular structures, the lumen is filled with nonpolarized vital cells. Madin-Darby canine kidney (MDCK) cells are an epithelial cell type commonly used as a model for cell polarization. Its-src-transformed variants are useful model systems for analyzing the changes in cell morphology, and they play a role in src-induced malignant transformation. This study investigates src-transformed cells in 3D cell cultures as a model for malignant transformation. The following questions were posed. Firstly: What is the role of the composition and stiffness of the extracellular matrix (ECM) on the polarization and transformation of ts v-src MDCK cells in 3D cell cultures? Secondly: How do the culture conditions affect gene expression? What is the effect of v-src transformation in 2D and in 3D cell models? How does the shift from 2D to 3D affect cell polarity and gene expression? Thirdly: What is the role of survivin and its regulator phosphatase and tensin homolog protein (PTEN) in cell polarization and transformation, and in determining cell fate? How does their expression correlate with impaired mitochondrial function in transformed cells? In order to answer the above questions, novel methods of culturing and monitoring cells had to be created: novel 3D methods of culturing epithelial cells were engineered, enabling real time monitoring of a polarization and transformation process, and functional testing of 3D cell cultures. Novel 3D cell culture models and imaging techniques were created for the study. Attention was focused especially on confocal microscopy and live-cell imaging. Src-transformation disturbed the polarization of the epithelium by disrupting cell adhesion, and sensitized the cells to their environment. With active src, the morphology of the cell cluster depended on the composition and stiffness of the matrix. Gene expression studies revealed a broader impact of src transformation than mere continuous activity of src-kinase. In 2D cultures, src transformation altered the expression of immunological, actin cytoskeleton and extracellular matrix (ECM). In 3D, the genes regulating cell division, inhibition of apoptosis, cell metabolism, mitochondrial function, actin cytoskeleton and mechano-sensing proteins were altered. Surprisingly, changing the culture conditions from 2D to 3D affected also gene expression considerably. The microarray hit survivin, an inhibitor of apoptosis, played a crucial role in the survival and proliferation of src-transformed cells.

Interactions of tris buffer with nucleotides: the crystal structure of tris(hydroxymethyl)methylammonium adenosine 5'-diphosphate dihydrate

The crystal and molecular structures of the Tris salt of adenosine 5'-diphosphate were determined from X-ray diffraction data. The crystals are monoclinic, space P21, and Z = 2 with a=9.198 (2) A, b=6.894 (1) A, c=18.440 (4) A, and beta = 92.55 (2) degrees. Intensity data were collected on an automated diffractometer. The structure was solved by the heavy-atom technique and refined by least squares to R = 0.047. The ADP molecule adopts a folded conformation. The conformation about the glycosidic bond is anti. The conformation of the ribose ring is close to a perfect C(2')-endo-C-(3')-exo puckering. The conformation about C(4')-C(5') is gauche-gauche, similar to other nucleotide structures. The pyrophosphate chain displays a nearly eclipsed geometry when viewed down the P-P vector, unlike the staggered conformation observed in crystal structures of other pyrophosphates. The less favorable eclipsed conformation probably results from the observed association of Tris molecules with the polar diphosphate chain through electrostatic interactions and hydrogen bonds. Such interactions may play an important role in Tris-buffered aqueous solutions of nucleotides and metal ions.

Temperature dependent free energy surface of polymer folding from equilibrium and quench studies

Langevin dynamics simulation studies have been employed to calculate the temperature dependent free energy surface and folding characteristics of a 500 monomer long linear alkane (polyethylene) chain with a realistic interaction potential. Both equilibrium and temperature quench simulation studies have been carried out. Using the shape anisotropy parameter (S) of the folded molecule as the order parameter, we find a weakly first order phase transition between the high-temperature molten globule and low-temperature rodlike crystalline states separated by a small barrier of the order of k(B)T. Near the melting temperature (580 K), we observe an intriguing intermittent fluctuation with pronounced ``1/f noise characteristics'' between these two states with large difference in shape and structure. We have also studied the possibilities of different pathways of folding to states much below the melting point. At 300 K starting from the all-trans linear configuration, the chain folds stepwise into a very regular fourfold crystallite with very high shape anisotropy. Whereas, when quenched from a high temperature (900 K) random coil regime, we identify a two step transition from the random coiled state to a molten globulelike state and, further, to a anisotropic rodlike state. The trajectory reveals an interesting coupling between the two order parameters, namely, radius of gyration (R-g) and the shape anisotropy parameter (S). The rodlike final state of the quench trajectory is characterized by lower shape anisotropy parameter and significantly larger number of gauche defects as compared to the final state obtained through equilibrium simulation starting from all-trans linear chain. The quench study shows indication of a nucleationlike pathway from the molten globule to the rodlike state involving an underlying rugged energy landscape. (C) 2010 American Institute of Physics. doi:10.1063/1.3509398]

Conformational Polymorphism in Telomeric Structures: Loop Orientation and Interloop Pairing in d(G4TnG4),

Sequence repeats constituting the telomeric regions of chromosomes are known to adopt a variety of unusual structures, consisting of a G tetraplex stem and short stretches of thymines or thymines and adenines forming loops over the stem. Detailed model building and molecular mechanics studies have been carried out for these telomeric sequences to elucidate different types of loop orientations and possible conformations of thymines in the loop. The model building studies indicate that a minimum of two thymines have to be interspersed between guanine stretches to form folded-back structures with loops across adjacent strands in a G tetraplex (both over the small as well as large groove), while the minimum number of thymines required to build a loop across the diagonal strands in a G tetraplex is three. For two repeat sequences, these hairpins, resulting from different types of folding, can dimerize in three distinct ways-i.e., with loops across adjacent strands and on same side, with loops across adjacent strands and on opposite sides, and with loops across diagonal strands and on opposite sides-to form hairpin dimer structures. Energy minimization studies indicate that all possible hairpin dimers have very similar total energy values, though different structures are stabilized by different types of interactions. When the two loops are on the same side, in the hairpin dimer structures of d(G(4)T(n)G(4)), the thymines form favorably stacked tetrads in the loop region and there is interloop hydrogen bonding involving two hydrogen bonds for each thymine-thymine pair. Our molecular mechanics calculations on various folded-back as well as parallel tetraplex structures of these telomeric sequences provide a theoretical rationale for the experimentally observed feature that the presence of intervening thymine stretches stabilizes folded-back structures, while isolated stretches of guanines adopt a parallel tetraplex structure

Conformational Polymorphism in G-tetraplex Structures: Strand Reversal by Base Flipover or Sugar Flipover

Guanine rich sequences adopt a variety of four stranded structures, which differ in strand orientation and conformation about the glycosidic bond even though they are all stabilised by Hoogsteen hydrogen bonded guanine tetrads. Detailed model building and molecular mechanics calculations have been carried out to investigate various possible conformations of guanines along a strand and different possible orientations of guanine strands in a G-tetraplex structure. It is found that for an oligo G stretch per se, a parallel four stranded structure with all guanines in anti conformation is favoured over other possible tetraplex structures. Hence an alternating syn-anti arrangement of guanines along a strand is likely to occur only in folded back tetraplex structures with antiparallel G strands. Our study provides a theoretical rationale for the observed alternation of glycosidic conformation and the inverted stacking arrangement arising from base flipover, in antiparallel G-tetraplex structures and also highlights the various structural features arising due to different types of strand orientations. The molecular mechanics calculations help in elucidating the various interactions which stabilize different G-tetraplex structures and indicate that screening of phosphate charge by counterions could have a dramatic effect on groove width in these four stranded structures.

Conformational polymorphism in G-tetraplex structures: strand reversal by base flipover or sugar flipover

Guanlne rich sequences adopt a variety of four stranded structures, which differ in strand orientation and conformation about the glycosldic bond even though they are all stabilised by Hoogsteen hydrogen bonded guanlne tetrads. Detailed model building and molecular mechanics calculations have been carried out to investigate various possible conformations of guanlnes along a strand and different possible orientations of guanlne strands In a G-tetraplex structure. It is found that for an ollgo G stretch per se, a parallel four stranded structure with all guanines In anti conformation is favoured over other possible tetraplex structures. Hence an alternating syn-anti arrangement of guanlnes along a strand is likely to occur only in folded back tetraplex structures with antiparallel G strands. Our study provides a theoretical rationale for the observed alternation of glycosldic conformation and the inverted stacking arrangement arising from base filpover, In antlparallel G-tetraplex structures and also highlights the various structural features arising due to different types of strand orientations. The molecular mechanics calculations help in elucidating the various interactions which stabilize different G-tetraplex structures and indicate that screening of phosphate charge by counterions could have a dramatic effect on groove width in these four stranded structures.

Folding of a Donor-Containing Ionene by Intercalation with an Acceptor

Cationic ionenes that bear electron-rich 1,5-dialkoxynaphthalene (DAN) units within the alkylene segment were allowed to interact with different types of electron-deficient, acceptor-containing molecules in an effort to realize intercalation-induced folding of the ionenes; the collapse of the chains was expected to occur in such a way that the donor and acceptor units become arranged in an alternating fashion. Several acceptor-bearing molecules were prepared by the derivatization of pyromellitic dianhydride and naphthalene tetracarboxylic dianhydride with two different oligoethylene glycol monomethyl ether monoamines. This yielded acceptor molecules with different water solubility and allowed the examination of solvophobic effects in the folding process. UV/Vis spectroscopic studies were carried out by using a 1:1 mixture of the DAN-ionenes and different acceptor molecules in water/DMSO solvent mixtures. The intensity of the charge-transfer (CT) band was seen to increase with the water content in the solvent mixture, thereby suggesting that the intercalation is indeed aided by solvophobic effects. The naphthalene diimide (NDI) bearing acceptor molecules consistently formed significantly stronger CT complexes when compared to the pyromellitic diimide (PDI) bearing acceptor molecules, which is a reflection of the stronger pi-stacking tendency of the former. AFM studies of drop-cast films of different ionene-acceptor combinations revealed that compact folded structures are formed most effectively under conditions in which the strongest CT complex is formed.

Hybrid Polypeptides: Gabapentin as a Stereochemically Constrained gamma-Amino Acid Residue

The design of folded structures in peptides containing the higher homologues of alpha-amino acid residues requires the restriction of the range of local conformational choices In alpha-amino acids stereochemically constrained residues like alpha,alpha-dialkylated residue, aminoisobutyric acid (Aib), and D-Proline ((D)Pro) have proved extremely useful in the design of helices and hairpins in short peptides Extending this approach, backbone substitution and cyclization are anticipated to bc useful in generating conformationally constrained beta- and gamma-residues This brief review provides a survey of work on hybrid peptide sequences concerning the conformationally constrained gamma-amino acid residue 1-aminomethyl cyclohexane acetic acid, gabapentin (Gpn) This achiral, beta,beta-disubstituted, gamma-residue strongly favors gauche-gauche conformations about the C-alpha-C-beta (0(2)) and C-alpha-C-gamma (0(1)) bonds, facilitating local folding The Gpn residue can adopt both C-7 (NH1 -> CO1) and C-9 (CO1 (I)<- NH1+I) hydrogen bonds which are analogous to the C-5 and C7 (gamma-turn) conformations at alpha-residues In conjunction with adjacent residues, Gpn may be used in ay and gamma alpha segments to generate C-12 hydrogen bonded conformations which may be considered as expanded analogs of conventional beta-turns The structural characterization of C-12 helices, C-12/C-10 helices with mixed hydrogen bond directionalities and beta-hairpins incorporating Gpn residues at the turn segment is illustrated (C) 2010 Wiley Periodicals, Inc Biopolymers (Pept Sci) 94 733-741 2010

Structure of N-tert-butyloxycarbonyl-[alpha]-aminoisobutyryl-DL-pipecolyl-[alpha]-aminoisobutyric acid methyl ester

C20H35N3O6 (Boc-Aib-DL-Pip-Aib-OMe, Boc = tert-butyloxycarbonyl, Aib = alpha-aminoisobutyric acid, Pip = pipecolic acid, OMe = methoxy), M(r) = 413.5, monoclinic, P2(1)/c, a = 18.055 (3), b = 15.048 (3), c = 17.173 (3) angstrom, beta = 91.7 (1)-degrees, V = 4663.8 (9) angstrom3, Z = 8, D(m) = 1.16, D(x) = 1.178 Mg m-3, lambda(Mo Kalpha) = 0.71069 angstrom, mu = 0.081 mm-1, F(000) = 1792, T = 297 K. The final R value for 4925 [I greater-than-or-equal-to 3sigma(I)] reflections is 0.065 (wR = 0.067). The peptide backbone of the two independent molecules in the asymmetric unit is folded at the -Aib-Pip- sequence to form a type-I (I') beta-bend stabilized by a 1 <-- 4 intramolecular N-H...O=C hydrogen bond between the Aib(3) peptide N-H and Boc urethane C=O groups.

Chain folding and A:T pairing in human telomeric DNA: a model-building and molecular dynamics study

The various types of chain folding and possible intraloop as well as interloop base pairing in human telomeric DNA containing d(TTAG(3)) repeats have been investigated by model-building, molecular mechanics, and molecular dynamics techniques. Model-building and molecular mechanics studies indicate that it is possible to build a variety of energetically favorable folded-back structures with the two TTA loops on same side and the 5' end thymines in the two loops forming TATA tetrads involving a number of different intraloop as well as interloop A:T pairing schemes. In these folded-back structures, although both intraloop and interloop Watson-Crick pairing is feasible, no structure is possible with interloop Hoogsteen pairing. MD studies of representative structures indicate that the guanine-tetraplex stem is very rigid and, while the loop regions are relatively much more flexible, most of the hydrogen bonds remain intact throughout the 350-ps in vacuo simulation. The various possible TTA loop structures, although they are energetically similar, have characteristic inter proton distances, which could give rise to unique cross-peaks in two-dimensional nuclear Overhauser effect spectroscopy (NOESY) experiments. These folded-back structures with A:T pairings in the loop region help in rationalizing the data from chemical probing and other biochemical studies on human telomeric DNA.

Crystal and molecular structure of Boc-Phe-Val-OMe; comparison of the peptide conformation with its dehydro analogue

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P2(1)2(1)2(1), a = 11.843(2), b = 21.493(4), c = 26.676(4)Angstrom and V = 6790 Angstrom(3). Data were collected on a CAD4 diffractometer using MoK2 radiation (lambda = 0.7107 Angstrom) up to Bragg angle theta = 26 degrees. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystallographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val(2) residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-Delta Phe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-Delta Phe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the Delta Phe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-Delta Phe-Val-OH are conformationally different, they exhibit similar packing patterns in the solid state. (C) Munksgaard 1995.

Refolding of riboflavin carrier protein as probed by biochemical and immunological parameters

The unfolding of the chicken egg white riboflavin carrier protein by disulfide reduction with dithiothreitol led to aggregation with concomitant loss of ligand binding characteristics and the capacity to interact with six monoclonal antibodies directed against surface-exposed discontinuous epitopes. The reduced protein could, however, bind to a monoclonal antibody recognizing sequential epitope. Under optimal conditions of protein refolding, the vitamin carrier protein regained its folded structure with high efficiency with simultaneous complete restoration of hydrophobic flavin binding site as well as the epitopic conformations exposed at the surface in a manner comparable to its native form.