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Biomedical subjects

M Sundaralingam

Publications and source records attributed to M Sundaralingam.

At least 55 records · Page 3Linked to original sources
PubMed1997

Correlation of hydrophobicity and packing in A-DNA oligonucleotides.

A-DNA oligomers pack in a slanted fashion with the terminal base pairs abutting into the minor groove of neighboring molecules unlike the other forms of DNA which pack by vertically stacking one over the other into helical columns. To explain the differences in packing we have advanced a hypothesis that the orientation of the sugar-phosphate backbone is different in A-DNA from that in the other forms of DNA, mainly due to the differences in the sugar puckering.

DNA↗
PubMed1996

Phospholipase A2 engineering. Deletion of the C-terminus segment changes substrate specificity and uncouples calcium and substrate binding at the zwitterionic interface.

It has been suggested [Dijkstra, B. W., Drenth, J., & Kalk, K. H. (1981) Nature 289, 604-606] that the interfacial binding site of phospholipase A2 (PLA2) involves a large number of residues, including a cluster at the N-terminus and another cluster at the C-terminus. The approaches of multiple mutation and deletion were used to test the roles of the C-terminal residues of bovine pancreatic PLA2 overexpressed in Escherichia coli. A double mutant K120A/K121A and a deletion mutant delta 115-123/ C27A were constructed, and structural and functional analyses were performed on both mutants. The double mutant showed little perturbation in the global structure on the basis of proton NMR and X-ray crystallographic analyses. The proton NMR analysis of the deletion mutant suggested that a few residues at the active site, the hydrophobic channel, and the calcium binding loop are perturbed, but the global conformation is not changed. The mutants were then characterized for catalytic and binding properties by use of various kinetic and spectroscopic methods. The double mutant behaved in a manner similar to that of the wild type (WT) PLA2 in every property examined. The deletion mutant was found to show an interesting change of substrate specificity. The kcat,app of the zwitterionic DC8PC micelles but not the anionic DC8PM micelles decreased by a factor of > 100; however, the activity of DC8PC was restored upon addition of 4 M NaCl. The results of fluorescence spectroscopic studies indicate that the deletion mutant behaves in a manner similar to that of WT in the binding to anionic vesicles and to zwitterionic neutral diluent. Thus, the binding affinity of the enzyme to the interface (the E to E* step) should not be the main cause for the change in substrate specificity. The cause lies at least partially in the binding of substrate or inhibitor to the active site of the enzyme at the interface, i.e., the E* to E*L step, as revealed by the results of equilibrium binding studies. The equilibrium dissociation constants of ligands are generally higher for the deletion mutant (relative to WT) at the zwitterionic interface but not at the anionic interface. The cause for the low affinity of an active site-directed ligand to the active site at the zwitterionic interface could be related to the inability of Ca2+ to enhance ligand binding for the deletion mutant. This is in contrast to the WT PLA2 for which Ca2+ binding enhances binding of the substrate to the active site. Overall, the structural and functional perturbations caused by deleting the C-terminal segment are modest, but the changes in substrate specificity and the uncoupling between substrate and calcium binding are interesting and significant.

Amino Acid Sequence↗
PubMed1996

Structure of d(CCCTAGGG): comparison with nine isomorphous octamer sequences reveals four distinct patterns of sequence-dependent intermolecular interactions.

The self-complementary deoxyoctanuceotide d(CCCTAGGG) crystallizes as an A-type double helix in the space group P4(3)2(1)2, a = b = 42.22 and c = 24.90 A, with one strand per asymmetric unit. Using 1533 unique reflections at 1.9 A and I > 2sigma, the structure was solved by molecular placement and refined to a final R value of 16.4%. This structure is isomorphous with nine other tetragonal A-DNA octamers, possessing a central pyrimidine/purine step that is fully extended along the backbone with trans, trans conformations around the C4'-C5' and O5'-P bonds. A structural water, sandwiched between the pi-cloud of the terminal guanine and the N3 atom of G7 in the adjacent duplex, stabilizes an intermolecular base triplet with one hydrogen bond between the terminal cytosine and G6 of an adjacent duplex. Comparative analysis of this structure with the isomorphous A-DNA octamers reveals the importance of base sequence and minor-groove hydration in intermolecular interactions. The minor grooves, which provide both hydrophobic and polar interactions, allow for four patterns of sequence-dependent binding involving interduplex base triplets in which the third base is bonded through a single hydrogen bond. A conserved water molecule appears to be crucial in the stabilization of these intermolecular interactions, which resemble specific recognition motifs found in the crystal structures of the TATA-box/TBP protein complex.

Journal Article↗
PubMed1996

X-ray structures of Mn, Cd and Tb metal complexes of troponin C.

The crystal structures of three metal complexes of troponin C (TnC) have been determined and refined where the two occupied structural Ca(2+) sites in the C domain have been substituted by Mn(2+), Cd(2+) and Tb(3+). The X-ray intensity data were collected to 2.1, 1.8 and 1.8 A resolution, respectively, on the three metal complexes, which are isomorphous with Ca-TnC. The three complexes have r.m.s. deviations of 0.27, 0.25 and 0.35 A, respectively, for all protein atoms, from Ca-TnC. Irrespective of the charge on the metal (+2 or +3), the occupied sites 3 and 4 exhibit a distorted pentagonal bipyramidal coordination, like Ca-TnC, with seven ligands, six from the 12-residue binding loop and the seventh from a water molecule. Mn(2+) at site 4 seems to display a longer distance to one of the carboxyl bidentate ligands representing an intermediate coordination simulating the six-coordinate Mg(2+). The carboxyl O atoms of the bidentate Glu12 are displaced on the side of the equatorial plane passing through the remaining three ligands with one O atom closer to the plane (Delta of 0.11 to 0.76 A) than the other (Delta of 0.93 to 1.38 A). The two axial ligands are an aspartic carboxyl O atom and a water molecule. The metal is displaced (0.18 to 0.56 A) towards the water facing the water channel.

Journal Article↗
PubMed1996

Structure of the purine-pyrimidine alternating RNA double helix, r(GUAUAUA)d(C), with a 3'-terminal deoxy residue.

The crystal structure of the purine-pyrimidine alternating octameric RNA helix, r(GUAUAUA)d(C), carrying a 3'-terminal deoxycytidine residue, has been determined at 2.2 A resolution. The molecule crystallizes in the rhombohedral space group R3 (hexagonal cell constants: a = b = 43.07,c = 59.36 A;alpha = beta = 90,gamma = 120 degrees )with one duplex in an asymmetric unit. The structure was solved by molecular replacement and refined with 83 and 2/3 solvent molecules and 2/3 sodium ions to a final R factor of 15.6% using 1775 reflections (86%). The duplexes are approximately linear, their global helix axes are inclined by 10 degrees with respect to the 3(2)-screw axes, and they are stacked on top of each other in a head-to-tail fashion. The twist between the junction base pairs of the stacked duplexes is negligible resulting in a discontinuity of the helix backbones and grooves. The sodium ions on the threefold axis play a significant role in the organization of the packing network. The helical parameters, particularly the twist and the roll, of this alternating sequence are in accord with Calladine's rules. Almost all the 2'-hydroxyl groups are involved in specific hydrogen-bonding interactions, either directly to the sugar ring oxygens O4' on the 3' side, or, through water bridges, to the sugars, phosphates, or bases. This hydrogen bonding of the 2'-hydroxyl groups restrains the conformation of the sugar-phosphate backbone and the glycosidic torsion angles of this RNA fragment. The lack of intermolecular packing contacts in the grooves provides a clear picture of the groove solvation.

Journal Article↗
PubMed1996

RNA - synthesis, purification and crystallization.

Protocols for the routine chemical synthesis and purification of milligram quantities of RNA and DNA-RNA chimeras meeting the demands of X-ray crystallography are described. An efficient screening protocol to test the crystallizability of the molecules and the optimization of the crystallization conditions are presented, so as to allow reproduction by others. Essentially the same crystallization conditions as for DNA oligomers can be employed for RNA crystallization. Specific examples involving alternating octamers, G/C-rich decamers, sequences with overhangs, and drug complexes of chimeras are discussed. Success of the methods is attested by the crystals obtained which diffract to high resolution.

Journal Article↗
PubMed1996

Studies of monoclinic hen egg-white lysozyme. IV. X-ray refinement at 1.8 A resolution and a comparison of the variable regions in the polymorphic forms.

Monoclinic crystals of hen egg-white lysozyme (E.C. 3.2.1.17, HEL) grown at low pH in the presence of NaNO(3) belong to space group P2(1) with unit-cell dimensions, a = 28.0, b = 62.5, c = 60.9 A and beta= 90.8 degrees with two molecules in the asymmetric unit. 1.8 A resolution intensity data, collected on a CAD-4 diffractometer, contained 17 524 reflections with F > 3sigma (93% complete). Our earlier preliminary 1.8 A model was refitted and refined using X-PLOR to an R value of 0.189. The deviations in the model from ideal geometry are 0.013 A in bond lengths and 2.8 degrees in bond angles. The r.m.s. deviation in the backbone atoms between the two molecules is 0.42 A. A comparison of HEL in different polymorphic crystal forms reveals that the prominent structural variability among them resides in two exposed regions 45-50 and 65-73 which are also regions of lattice contacts.

Journal Article↗
PubMed1996

Crystal structure of the self-complementary 5'-purine start decamer d(GCGCGCGCGC) in the Z-DNA conformation. I.

Alternating self-complementary oligonucleotides starting with a 5'-pyrimidine usually form left-handed Z-DNA; however, with a 5'-purine start sequence they form the right-handed A-DNA. Here we report the crystal structure of the decamer d(GCGCGCGCGC) with a 5'-purine start in the Z-DNA form. The decamer crystallizes in the hexagonal space group P6(5)22, unit cell dimensions a = b = 18.08 and c = 43.10 A, with one of the following four dinucleotide diphosphates in the asymmetric unit: d(pGpC)/d(GpCp)/d(pCpG)/d(CpGp). The molecular replacement method, starting with d(pGpC) of the isomorphous Z-DNA hexamer d(araC-dG)3 without the 2'-OH group of arabinose, was used in the structure analysis. The method gave the solution only after the sugar-phosphate conformation of the GpC step was manipulated. The refinement converged to a final R value of 18.6% for 340 unique reflections in the resolution range 8.0-1.9 A. A result of the sequence alternation is the alternation in the nucleotide conformation; guanosine is C3'-endo, syn, and cytidine is C2'-endo, anti. The CpG step phosphodiester conformation is the same as ZI or ZII, whereas that of the GpC step phosphodiester is "intermediate" in the sense that zeta (O3'-P bond) is the same as ZII but alpha (P-O5' bond) is the same as ZI. The duplexes generated from the dinucleotide asymmetric unit are stacked one on top of the other in the crystal to form an infinite pseudocontinuous helix. This renders it a quasi-polymerlike structure that has assumed the Z-DNA conformation further strengthened by the long inner Z-forming stretch d(CG)4. An interesting feature of the structure is the presence of water strings in both the major and the minor grooves. In the minor groove the cytosine carbonyl oxygen atoms of the GpC and CpG steps are cross-bridged by water molecules that are not themselves hydrogen bonded but are enclosed by the water rings in the mouth of the minor groove. In the major groove three independent water molecules form a zigzagging continuous water string that runs throughout the duplex.

Base Sequence↗
PubMed1996

Crystal structure of the self-complementary 5'-purine start decamer d(GCACGCGTGC) in the A-DNA conformation. II.

The crystal structure of the alternating 5'-purine start decamer d(GCGCGCGCGC) was found to be in the left-handed Z-DNA conformation. Inasmuch as the A.T base pair is known to resist Z-DNA formation, we substituted A.T base pairs in the dyad-related positions of the decamer duplex. The alternating self-complementary decamer d(GCACGCGTGC) crystallizes in a different hexagonal space group, P6(1)22, with very different unit cell dimensions a = b = 38.97 and c = 77.34 A compared with the all-G.C alternating decamer. The A.T-containing decamer has one strand in the asymmetric unit, and because it is isomorphous to some other A-DNA decamers it was considered also to be right-handed. The structure was refined, starting with the atomic coordinates of the A-DNA decamer d(GCGGGCCCGC), by use of 2491 unique reflections out to 1.9-A resolution. The refinement converged to an R value of 18.6% for a total of 202 nucleotide atoms and 32 water molecules. This research further demonstrates that A.T base pairs not only resist the formation of Z-DNA but can also assist the formation of A-DNA by switching the helix handedness when the oligomer starts with a 5'-purine; also, the length of the inner Z-DNA stretch (d(CG)n) is reduced from an octamer to a tetramer. It may be noted that these oligonucleotide properties are in crystals and not necessarily in solutions.

Base Sequence↗
PubMed1996

Crystal structure of the B-DNA hexamer d(CTCGAG): model for an A-to-B transition.

The crystal structure of the B-DNA hexamer d(CTCGAG) has been solved at 1.9 A resolution by iterative single isomorphous replacement, using the brominated derivative d(CG5BrCGAG), and refined to an R-factor of 18.6% for 120 nonhydrogen nucleic acid atoms and 32 water molecules. Although the central four base pairs form a typical B-form helix, several parameters suggest a transition to an A-like conformation at the termini. Based on this observation, a B-to-A transition was modeled, maintaining efficient base stacking across the junction. The wide minor groove (approximately 6.9 A) is reminiscent of that in the side-by-side double drug-DNA complexes and hosts a double spine of hydration. The global helix axes of the pseudo-continuous helices are at an acute angle of 60 degrees. The pseudocontinuous stacking is reinforced by the minor groove water structure extending between the two duplexes. The crossover point of two pairs of stacked duplexes is at the stacking junction, unlike that observed in the B-DNA decamers and dodecamers. This arrangement may have implications for the structure of a four-way DNA junction. The duplexes are arranged around a large (approximately 20 A diameter) channel centered on a 6(2) screw axis.

Base Sequence↗
PubMed1996

The structure of r(UUCGCG) has a 5'-UU-overhang exhibiting Hoogsteen-like trans U.U base pairs.

The crystal structure of the RNA fragment, 5'-r(UUCGCG)-3', has been determined at 1.4 A resolution by a combination of single isomorphous replacement and molecular search methods. The 3'-terminal CGCG portion of the hexamer engages in Watson-Crick hydrogen bonding while the 5'-terminal UU-overhang forms novel Hoogsteen-like U.U self-base pairs with the overhang of an adjacent duplex. The U.U pairs display a single conventional hydrogen bond between O4 (U1) and N3 (U8) and a CH-O hydrogen bond between C5-H (U1) and O4 (U8), through the Hoogsteen face of the pyrimidine base U1. This unusual arrangement of one of the bases results in a trans U.U pair on antiparallel strands in contrast to the usual cis base pairs. The structure emphasizes the pronounced polymorphism of U.U pairs and has implications for folding of RNA molecules.

Animals↗
PubMed1995

2',3'-Dideoxy-3'-C,2'-N-[(3R,5R)-5-ethoxy-carbonyl-2-methyl-1, 2-isoxazolidine]-ribothymidine.

The title compound, C15H21N3O7, is a thymidine derivative with a tetrahydroisoxazole (THI) ring fused to the sugar ring at the 2'- and 3'-C atoms. The thymine base is in an anti conformation [chi = -122.5 (3) degrees] while the ribose moiety has C2'-exo,C3'-endo (3T2) conformation with a highly flattened sugar ring [P = -5.8, tau m = 13.1 degrees]. The molecule contains a pseudo-twofold axis on the C2'-C3' bond with the ethyl ester group acting as the base and the N-methyl group acting as the 5'-hydroxyl group in a nucleoside. The THI ring fused to the furanose has a C3'-endo,C4'-exo (3T4) conformation [P = 4.18, tau m = 19.3 degrees]. The ethyl ester moiety is disordered with respect to two conformations, the population of the major form being 87.4 (8)% and that of the minor form 12.6 (8)%.

Antiviral Agents↗
PubMed1995

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, X-ray, and NMR.

The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

Amino Acid Sequence↗
PubMed1995

Structure of the trigonal form of recombinant oxidized flavodoxin from Anabaena 7120 at 1.40 A resolution.

The oxidized recombinant flavodoxin from the cyanobacterium Anabaena 7120 has been crystallized in a trigonal form. The recombinant protein has an identical primary structure to that purified directly from Anabaena, which functions as a substitute for ferredoxin in an iron-deficient environment for electron transfer from photosystem I to ferredoxin-NADP(+) reductase. X-ray data to 1.40 A were collected on a Siemens area detector. Of the 311 379 reflections collected, 36069 reflections were unique in space group P3(1)21 (a = 55.36, c = 102.59 A) with an R(merge) of 3.8%. The structure was solved by molecular replacement using coordinates from the wild-type monoclinic structure previously solved in this laboratory [Rao, Shaffie, Yu, Satyshur, Stockman & Markley (1992). Protein Sci. 1, 1413-1427]. The structure was refined with X-PLOR and SHELXL93 to a crystallographic R-factor of 13.9% for 32963 reflections with I> 2sigma(I). The final structure contains 2767 atoms including 31 flavin mononucleotide (FMN) atoms, 299 water molecules, and one sulfate ion. The protein is comprised of a central five-stranded beta-sheet surrounded by five helices and binds a single molecule of FMN at the C-terminus of the sheet. The trigonal protein structure and the crystal packing are compared with the monoclinic wild-type protein. Helix alpha3 in this structure is less distorted than in the monoclinic structure and shows additional hydrogen bonds in the N-terminal portion of the helix. The trigonal structure is extensively hydrogen bonded in three major areas with neighboring molecules compared with five regions in the monoclinic structure, but using significantly fewer hydrogen bonds to stabilize the lattice. There are several hydrogen bonds to the amide groups from water molecules several of which stabilize and extend the ends of the beta-sheet.

Journal Article↗
PubMed1995

X-ray structures of the B-DNA dodecamer d(CGCGTTAACGCG) with an inverted central tetranucleotide and its netropsin complex.

The crystal structures of the B-DNA dodecamer d(CGCGTTAACGCG) duplex (T2A2), with the inverted tetranucleotide core from the duplex d(CGCGAATTCGCG) [A2T2, Dickerson & Drew (1981). J. Mol. Biol. 149, 761-768], and its netropsin complex (T2A2-N) have been determined at 2.3 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), unit-cell dimensions of a = 25.7, b = 40.5 and c = 67.0 A, for T2A2 and a = 25.49, b = 40.87, c = 67.02 A for T2A2-N and are isomorphous with A2T2. The native T2A2 structure, with 70 water molecules had a final R value of 0.15 for 1522 reflections (F > 2sigma), while for the netropsin complex, with 87 water molecules, the R value was 0.16 for 2420 reflections. In T2A2, a discontinuous string of zig-zagging water molecules hydrate the narrow A.T minor groove. In T2A2-N, netropsin binds in one orientation in the minor groove, covering the TTAA central region, by displacing the string of waters, forming the majority of hydrogen bonds with DNA atoms in one strand, and causing very little perturbation of the native structure. The helical twist angle in T2A2 is largest at the duplex center, corresponding to the cleavage site by the restriction enzymes HpaI and HincII. The sequence inversion AATT-->TTAA of the tetranucleotide at the center of the molecule results in a different path for the local helix axis in T2A2 and A2T2 but the overall bending is similar in both cases.

Journal Article↗
PubMed1995

New crystal structures of nucleic acids and their complexes.

In the past year, X-ray crystallographic studies of representatives of all nucleic acid structural types have been reported. Among the most interesting structures are the parallel DNA tetraplex formed by d(TGGGGT), the four-stranded structure formed by d(CCCT) and a double drug bound side by side in an antiparallel orientation to the minor groove of a B-DNA. Certainly, the structure that has received most attention is that of the first complex of a ribozyme with an inhibitor DNA.

Animals↗
PubMed1995

Crystal structure of the A-DNA decamer d(CCIGGCCm5CGG) at 1.6 A showing the unexpected wobble I.m5C base pair.

The crystal structure of the self-complementary decamer d(CCIGGCCm5CGG), where I and m5C replace A and T, respectively, in the Watson-Crick B-DNA decamer d(CCAGGCCTGG) is in the A-DNA conformation. Furthermore, the A-DNA duplex exhibits the unexpected wobble I.m5C+ base pairs with N3 of 5-methylcytosine protonated. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with a = 25.02, b = 44.95 and c = 47.62 A with one DNA duplex in the asymmetric unit. Intensity data were collected on our Siemens area detector to 1.6 A resolution. The structure was solved by the molecular placement method starting with a model from an isomorphous structure. The refinement gave a final R value of 16.3% for 404 DNA atoms and 104 water molecules using 5119 reflections. The hydration of the I.m5C+ wobble base pairs in the major groove stabilizes them as in the G.T/G.U wobbles. A comparison with the Watson-Crick base pairs of another isomorphous structure d(GCGGGCCCGC) reveals that the wobble base pairs are better described by a rotation of the individual nucleotide units around their centers of gravity. This is in contrast to the earlier description of translation of the bases into the grooves. The exposed N4 amino group of 5-methylcytosine in the wobble base pair provides a rationale for its deamination to thymine.

5-Methylcytosine↗