Comparative structural analysis of cytidine, ethenocytidine and their protonated salts III. 1H, 13C and 15N NMR studies at natural isotope abundance

The 1H, 13C, 15N NMR spectra of cytidine /Cyd/, ethenocytidine /epsilon Cyd/ and their hydrochlorides /Cyd X HC1/ and /epsilon Cyd X HC1/ have been analysed to compare structural differences observed in solution with those existing in the crystalline state. The effects of ethenobridging and protonation of the hertero-aromatic base on the intramolecular stereochemistry, intermolecular interactions and electronic structure of the whole molecule are discussed on the basis of the NMR studies in DMSO solutions. Particular interest is devoted to the discussion of the conformation of the ribose ring, the presence of the intramolecular C-5'-0...H-6-C hydrogen bond, unambiguous assignment of the site of protonation, the mechanism of the 5C-H deuterium exchange in Cyd X HC1, and the intermolecular interactions in solution.     

Comparative structural analysis of cytidine, ethenocytidine and their protonated salts. II. IR spectral studies.

The IR spectra of crystalline cytidine (Cyd), ethenocytidine (epsilon Cyd), and their hydrochlorides (Cyd-Hcl and epsilon CyD-HCl) have been analyzed to determine the spectroscopic manifestations of the structural differences that were previously established for these nucleosides from X-ray studies. O,N-Deuteration of the samples turned out to be a successful approach to obtaining interpretable spectra. The analysis was carried out in three frequency ranges: (i) The 2600-1900 cm-1 range originating from the vO-D and VN-D vibrations. All intermolecular hydrogen bonds could be recognized here. The positions of the individual vO-D (vN-D) bands were correlated with the geometrical delta HB parameters presenting the strengths of hydrogen bonds in which these groups act as donors (ii) The 1750-1500 cm-1 region originating from the stretching vibrations of double bonds. All absorption bands in this region were interpreted in terms of electronic structures of the base fragments. (iii) The region of the C-H stretching vibrations of the base fragments (3200-3000 cm-1) and sugar moieties (3000-2800 cm-1). The Csp2-H vibrations also reflect the electronic structures of the base fragments, whereas the vCsp-H frequencies seem to be sensitive to etheno-bridging and to the presence of an intramolecular C6-H...05' hydrogen bond.     

Calculations on crystal packing of a flexible molecule, Leu-enkephalin

Crystal packing calculations have been carried out on a substantial number of conformations of Leu-enkephalin; namely, those obtained both from crystal structures and from energy minimizations on isolated molecules, and with and without waters of crystallization. The known crystal structures represent the most energetically stable packings found. The conformations of the enkephalin molecules in the crystal are not the most stable for an isolated molecule; i.e. intermolecular interactions force the isolated molecule to change conformation in order to achieve a small packing volume and an optimal packing energy in the crystal. It is found that the packing energy of an enkephalin molecule is a reasonably smooth function of its molecular volume in the unit cell, if structures with intermolecular hydrogen bonding are excluded, and is substantially independent of other details of the molecular conformation or of the crystal packing. Hydrogen bonding provides additional stabilization of the crystal structure, and would likely permit crystallization of the system if it is sufficiently dense. Solvent molecules further stabilize the structure when they can also provide intermolecular hydrogen bonds.     

Photochemistry and photobiology of 5-azacytidine: effect of repair on photostabilization of Escherichia coli.

The photochemical stability of the anomalous nucleic acid base 5-azacytidine (z5Cyd) on irradiation at 254 nm is by about one order of magnitude less than that of cytidine (Cyd). Contrary to the photochemical behaviour, incorporation of z5Cyd into the nucleic acids of E. coli strains SR 20 (uvr+ rec+), SR 74 (uvr+ rec-) and SR 22 (uvr- rec+) produced a higher resistance to UV light. Only the SR 73 (uvr- rec-) strain was shown to have an increased UV sensitivity. This latter finding is in accord with the photochemical properties of z5Cyd. The results led to the conclusion that excision and recombination repair processes contribute to the observable protective effect. The fact that inhibition of excission repair by caffeine or proflavine of the mutant uvr+ rec- changes protection into sensitization supports this idea.     

Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking,molecular dynamics,and charge density analysis

Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer’s disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.     

Structure of crambin in solution, crystal and in the trajectories of molecular dynamics simulations

The mechanisms of the three-dimensional crambin structure alterations in the crystalline environments and in the trajectories of the molecular dynamics simulations in the vacuum and crystal surroundings have been analyzed. In the crystalline state and in the solution the partial regrouping of remote intramolecular packing contacts, involved in the formation and stabilization of the tertiary structure of the crambin molecule, occurs in NMR structures. In the crystalline state it is initiated by the formation of the intermolecular contacts, the conformational influence of its appearance is distributed over the structure. The changes of the conformations and positions of the residues of the loop segments, where the intermolecular contacts of the crystal surroundings are preferably concentrated, are most observable. Under the influence of these contacts the principal change of the regular secondary structure of crambin is taking place: extension of the two-strand β structure to the three-strand structure with the participation of the single last residue N46 of the C-terminal loop. In comparison with the C-terminal loop the more profound changes are observed in the conformation and the atomic positions of the backbone atoms and in the solvent accessibility of the residues of the interhelical loop. In the solution of the ensemble of the 8 NMR structures relative accessibility to the solvent differs more noticeably also in the region of the loop segments and rather markedly in the interhelical loop. In the crambin cryogenic crystal structures the positions of the atoms of the backbone and/or side chain of 14–18 of 46 residues are discretely disordered. The disorganizations of at least 8 of 14 residues occur directly in the regions of the intermolecular contacts and another 5 residues are disordered indirectly through the intramolecular contacts with the residues of the intermolecular contacts. Upon the molecular dynamics simulation in the vacuum surrounding as in the solution of the crystalline structure of crambin the essential changes of the backbone conformation are caused by the intermolecular contacts absence, but partly masked by the structure changes owing to the nonpolar H atoms absence on the simulated structure. The intermolecular contact absence is partly manifested upon the molecular dynamics simulation of the crambin crystal with one protein molecule. Compared to the crystal structure the lengths of the interpeptide hydrogen bonds and other interresidue contacts in an average solution NMR structure are somewhat shorter and accordingly the energy of the interpeptide hydrogen bonds is better. This length shortening can occur at the stage of the refinement of the NMR structures of the crambin and other proteins by its energy minimizations in the vacuum surroundings and not exist in the solution protein structures.     

Effect of point mutations on 5.8S ribosomal ribonucleic acid secondary structure and the 5.8S--28S ribosomal ribonucleic acid junction

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.     

Crystallographic characterization of an exocyclic DNA adduct: 3,N4-etheno-2'-deoxycytidine in the dodecamer 5'-CGCGAATTepsilonCGCG-3'

Exocyclic DNA adducts are formed from metabolites of chemical carcinogens and have also been detected as endogenous lesions in human DNA. The exocyclic adduct 3,N(4)-etheno-2'-deoxycytidine (epsilon dC), positioned opposite deoxyguanosine in the B-form duplex of the dodecanucleotide d(CGCGAATTepsilonCGCG), has been crystallographically characterized at 1.8A resolution. This self-complementary oligomer crystallizes in space group P3(2)12, containing a single strand in the asymmetric unit. The crystal structure was solved by isomorphous replacement with the corresponding unmodified dodecamer structure. Exposure of both structures to identical crystal packing forces allows a detailed investigation of the influence of the exocyclic base adduct on the overall helical structure and local geometry. Structural changes are limited to the epsilon C:G and adjacent T:A and G:C base-pairs. The standard Watson-Crick base-pairing scheme, retained in the T:A and G:C base-pairs, is blocked by the etheno bridge in the epsilon C:G pair. In its place, a hydrogen bond involving O2 of epsilon C and N1 of G is present. Comparison with an epsilon dC-containing NMR structure confirms the general conformation reported for epsilon C:G, including the hydrogen bonding features. Superposition with the crystal structure of a DNA duplex containing a T:G wobble pair shows similar structural changes imposed by both mismatches. Evaluation of the hydration shell of the duplex with bond valence calculations reveals two sodium ions in the crystal.     

Hydration of Cytidine, 2′-Deoxycytidine and their Phosphate Salts in the Aqueous Solutions. A Molecular Dynamics Computer Simulation Study

Abstract

To compare the hydration pattern of the cytidine (Cyd) and 2′-deoxycytidine (dCyd) in the aqueous solutions at the level of microscopic interactions, Molecular Dynamics (MD) computer simulations have been undertaken. The results indicate that the hydration of the heterocyclic base moiety in cytidine and 2′-deoxycytidine has a hydrophobic character. None of the three potential Watson—;Crick base pair centres hydrogen bonds with the water molecules and the formation of something akin to a clathrate cage structure of water around base moieties of nucleosides in the aqueous solution is suggested. In contrast, the hydration of Cyd and dCyd sugar moieties shows a hydrophilic character and the three-dimensional networks of H-bonds involving all hydrophilic centres are formed differently around the ribose and 2′-deoxyribose. The sugar hydroxyl groups participate in the hydrogen bonding with water both as H-donor and as H-acceptor. Their donor-acceptor abilities have been evaluated and compared. The coordination numbers, the geometrical data of the first hydration shell, and the number of hydrogen bonds have been calculated. The changes in the pattern of hydration with the increased concentration of nucleosides and upon nucleoside protonation are discussed. The analysis of the pairwise interaction energies are also presented.     

Three-dimensional structure of the complexes of ribonuclease A with 2',5'-CpA and 3',5'-d(CpA) in aqueous solution, as obtained by NMR and restrained molecular dynamics.

The three-dimensional structure of the complexes of ribonuclease A with cytidyl-2',5'-adenosine (2',5'-CpA) and deoxycytidyl-3',5'-deoxyadenosine [3',5'-d(CpA)] in aqueous solution has been determined by 1H NMR methods in combination with restrained molecular dynamics calculations. Twenty-three intermolecular NOE cross-corrections for the 3',5'-d(CpA) complex and 19 for the 2',5'-CpA, together with about 1,000 intramolecular NOEs assigned for each complex, were translated into distance constraints and used in the calculation. No significant changes in the global structure of the enzyme occur upon complex formation. The side chains of His 12, Thr 45, His 119, and the amide backbone group of Phe 120 are involved directly in the binding of the ligands at the active site. The conformation of the two bases is anti in the two complexes, but differs from the crystal structure in the conformation of the two sugar rings in 3',5'-d(CpA), shown to be in the S-type region, as deduced from an analysis of couplings between the ribose protons. His 119 is found in the two complexes in only one conformation, corresponding to position A in the free protein. Side chains of Asn 67, Gln 69, Asn 71, and Glu 111 from transient hydrogen bonds with the adenine base, showing the existence of a pronounced flexibility of these enzyme side chains at the binding site of the downstream adenine. All other general features on the structures coincide clearly with those observed in the crystal state.     

Staggered molecular packing in crystals of a collagen-like peptide with a single charged pair

The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.     

Three-dimensional structures of bulge-containing DNA fragments.

The three-dimensional structure of a DNA tridecamer d(CGCAGAATTCGCG)2 containing bulged adenine bases was determined by single crystal X-ray diffraction methods, at 120 K, to 2.6 A resolution. The structure is a B-DNA type double helix with a single duplex in the asymmetric unit. One of the bulged adenine bases loops out from the double helix, while the other stacks in to it. This is in contrast to our preliminary finding, which indicated that both adenine bases were looped out. This revised model was confirmed by the use of a covalently bound heavy-atom derivative. The conformation of the looped-out bulge hardly disrupts base stacking interactions of the bases flanking it. This is achieved by the backbone making a "loop-the-loop" curve with the extra adenine flipping over with respect to the other nucleotides in the strand. The looped-out base intercalates into the stacked-in bulge site of a symmetrically related duplex. The looped-out and stacked-in bases form an A.A reversed Hoogsteen base-pair that stacks between the surrounding base-pairs, thus stabilizing both bulges. The double helix is frayed at one end with the two "melted" bases participating in intermolecular interactions. A related structure, of the same tridecamer, after soaking the crystals with proflavin, was determined to 3.2 A resolution. The main features of this B-DNA duplex are basically similar to the native tridecamer but differ in detail especially in the conformation of the bulged-out base. Accommodation of a large perturbation such as that described here with minimal disruption of the double helix shows both the flexibility and resiliency of the DNA molecule.     

Structural features and hydration of a dodecamer duplex containing two C.A mispairs.

X-ray diffraction techniques have been used to characterise the crystal and molecular structure of the deoxyoligomer d(C-G-C-A-A-A-T-T-C-G-C-G) at 2.5A resolution. The final R factor is 0.19 with the location of 78 solvent molecules. The oligomer crystallises in a B-DNA type conformation with two strands coiled about each other to produce a duplex. This double helix consists of four A.T and six G.C Watson-Crick base pairs and two C.A mispairs. The mismatched base pairs adopt a "wobble" type structure with the cytosine displaced laterally into the major groove, the adenine into the minor groove. We have proposed that the two close contacts observed in the C.A pairing represent two hydrogen bonds one of which results from protonation of adenine. The mispairs are accommodated in the double helix with small adjustments in the conformation of the sugar-phosphate backbone. Details of the backbone conformation, base stacking interactions, thermal parameters and the hydration are now presented and compared with those of the native oligomer d(C-G-C-G-A-A-T-T-C-G-C-G) and with variations of this sequence containing G.T and G.A mispairs.     

Interaction of Pd(II) glycyl--L-histidine complex with cytidine and GMP. Proton and carbon-13 nmr studies

1H and 13C nmr studies on the Pd(II)Gly-His complex interaction with cytidine and GMP have shown that the nucleoside binds the palladium complex via N3 nitrogen and the nucleotide binds that complex via N7 nitrogen. The analysis of the Cyd or GMP aromatic ring influence on the chemical shift of the H2 proton or the C2 carbon of imidazole ring has supported the earlier suggestions that nucleoside or nucleotide base and Pd(II) complex plane are almost perpendicular to each other. The Pd(II)Gly-His: Cyd or GMP ternary systems are easily decomposed already in weak basic solutions, which may suggest that the polymerization of Pd(II)Gly-His binary species might be the competitive process in the interactions with nucleosides or nucleotides.     

Structure of an 11-mer DNA duplex containing the carbocyclic nucleotide analog: 2'-deoxyaristeromycin

2'-deoxyaristeromycin (dAr) is a nucleoside analogue that is resistant to the action of DNA glycosylases. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of an 11-mer DNA containing a single dAr.T base pair at its center. Analysis of the spectra revealed the existence of a right-handed duplex in solution, stabilized by Watson-Crick hydrogen bonding and base-stacking interactions. The carbocyclic sugar adopted a C1'-exo conformation and sugars of the 3'-flanking base pair had puckers in the O4'-endo range. The dAr.T base pair was mildly propeller twisted, and the dAr analogue showed a positive roll with the 3'-flanking base. Our findings indicate that the observed resistance of dAr-containing oligodeoxynucleotides to the catalytic action of DNA glycosylases relates to its electronic properties rather than structure, and validate the use of dAr and related carbocyclic nucleoside analogues for biological and structure/function relationship studies.     

The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion.

BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) is a serpin that has a key role in the control of fibrinolysis through proteinase inhibition. PAI-1 also has a role in regulating cell adhesion processes relevant to tissue remodeling and metastasis; this role is mediated by its binding to the adhesive glycoprotein vitronectin rather than by proteinase inhibition. Active PAI-1 is metastable and spontaneously transforms to an inactive latent conformation. Previous attempts to crystallize the active conformation of PAI-1 have failed. RESULTS: The crystal structure of a stable quadruple mutant of PAI-1(Asn150-->His, Lys154-->Thr, Gln319-->Leu, Met354-->Ile) in its active conformation has been solved at a nominal 3 A resolution. In two of four independent molecules within the crystal, the flexible reactive center loop is unconstrained by crystal-packing contacts and is disordered. In the other two molecules, the reactive center loop forms intimate loop-sheet interactions with neighboring molecules, generating an infinite chain within the crystal. The overall conformation resembles that seen for other active inhibitory serpins. CONCLUSIONS: The structure clarifies the molecular basis of the stabilizing mutations and the reduced affinity of PAI-1, on cleavage or in the latent form, for vitronectin. The infinite chain of linked molecules also suggests a new mechanism for the serpin polymerization associated with certain diseases. The results support the concept that the reactive center loop of an active serpin is flexible and has no defined conformation in the absence of intermolecular contacts. The determination of the structure of the active form constitutes an essential step for the rational design of PAI-1 inhibitors.     

Application of Raman spectroscopy to biological macromolecules.

The Raman spectra of biological macromolecules arise from molecular vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 cm-1 and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a macromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the averge conformation of their backbone chain. In polynucleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 cm-1 for A conformation and 790 cm-1 for B conformation. Raman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raman spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.     

Conformational, receptor interaction and alanine scan studies of glucose-dependent insulinotropic polypeptide

Glucose-dependent insulinotropic polypeptide (GIP) is an insulinotropic incretin hormone that stimulates insulin secretion during a meal. GIP has glucose lowering abilities and hence is considered as a potential target molecule for type 2 diabetes therapy. In this article, we present the solution structure of GIP in membrane-mimicking environments by proton NMR spectroscopy and molecular modelling. GIP adopts an α-helical conformation between residues Phe(6)-Gly(31) and Ala(13)-Gln(29) for micellar and bicellar media, respectively. Previously we examined the effect of N-terminal Ala substitution in GIP, but here eight GIP analogues were synthesised by replacing individual residues within the central 8-18 region with alanine. These studies showed relatively minor changes in biological activity as assessed by insulin releasing potency. However, at higher concentration, GIP(Ala(16)), and GIP(Ala(18)) showed insulin secreting activity higher than the native GIP (P<0.01 to P<0.001) in cultured pancreatic BRIN-BD11 cells. Receptor interaction studies of the native GIP with the extracellular domain of its receptor were performed by using two different docking algorithms. At the optimised docking conformation, the complex was stabilised by the presence of hydrophobic interactions and intermolecular hydrogen bonding. Further, we have identified some potentially important additional C-terminal interactions of GIP with its N-terminal extracellular receptor domain.     

tRNA tertiary structure in solution as probed by the photochemically induced 8-13 cross-link.

The conformation of ten purified tRNAs from Escherichia Coli has been investigated by means of the photo-induced cross-linking of 4Srd8 and Cyd13, which is sensitive to the juxtaposition of the two bases. Three tRNAs photo-react abnormally slowly; tRNAPhe, tRNAMet/m a and tRNAVal/2; a comparison with normally reacting species suggests that base 47 (Urd or modified Urd) is involved in a tertiary interaction in Class I tRNAs with the triplet 8, 14, 28. The UGA suppressor tRNATrp photoreacts significantly slower than the wild type. Thus the single base change Gua 24 to Ade induces a conformational change that alters the rate constant for the cross-linking reaction.     

Determination of structural peculiarities of dexran,pullulan and gamma-irradiated pullulan by Fourier-transform IR spectroscopy

Deconvoluted IR-absorbance spectra of dextran, pullulan and gamma-irradiated pullulan were analyzed in order to find the most specific spectral peculiarities that allow one to obtain information about the structure and conformation of these macromolecules in solvents that exhibit different influences on the system of intra- and intermolecular interactions. The changes in intensity and width of the IR bands at about 1040, 1020 and, in the case of pullulan, also at 996 cm(-1), were related to changes in conformation and short-range interactions of the polysaccharides. Furthermore, certain bands within the 1200-900 cm(-1) region were considered as a characteristic for the type of glycosidic linkage. The results of the FTIR spectroscopy study allowed one to suggest a predominant cleavage of the alpha-(1-->4) linkages upon the radiation-chemical destruction of pullulan.