ABSTRACT
Interaction of ß-D-glucopyranuronic acid (GlcA), N-acetyl-ß-D-glucosamine (GlcNAc), N-acetyl-ß-D-galactosamine (GalNAc) and two natural decameric glycosaminoglycans, hyaluronic acid (HA) and Chondroitin (Ch) with carboxylated carbon nanotubes, were studied using molecular dynamics simulations in a condensed phase. The force field used for carbohydrates was the GLYCAM-06j version, while functionalized carbon nanotubes (fCNT) were described using version two of the general amber force field. We found a series of significant differences in carbohydrate-fCNT adsorption strength depending on the monosaccharide molecule and protonation state of surface carboxyl groups. GlcNAc and GalNAc reveal a strong adsorption on fCNT with deprotonated carboxyl groups, and a slightly weaker adsorption on the fCNT with protonated carboxyl groups. On the contrary, GlcA weakly adsorbs on fCNT. The change in protonation state of surface carboxyl groups leads to the reversal orientation of GlcNAc and GalNAc in reference to the fCNT surface, while GlcA is not sensitive to that factor. Adsorption of decameric oligomers on the surface of fCNT weakens with the increasing number of monosaccharide units. Chondroitin adsorbs weaker than hyaluronic acid and incorporation of four Ch molecules leads to partial detachment of them from the fCNT surface. The glycan-fCNT interactions are strong enough to alter the conformation of carbohydrate backbone; the corresponding conformational changes act toward a more intensive contact of glycan with the fCNT surface. Structural and energetic features of the adsorption process suggest the CH-π interaction-driven mechanism.
Subject(s)
Chondroitin , Nanotubes, Carbon , Glycosaminoglycans , Hyaluronic Acid , Molecular Dynamics Simulation , Nanotubes, Carbon/chemistry , Monosaccharides , Carboxylic AcidsABSTRACT
In this paper, we describe molecular dynamics simulation results of the interactions between four peptides (mTM10, mTM16, TM17 and KTM17) with micelles of dodecylphosphocholine (DPC) and dodecyl-ß-d-maltoside (DDM). These peptides represent three transmembrane fragments (TM10, 16 and 17) from the MSD1 and MSD2 membrane-spanning domains of an ABC membrane protein (hMRP1), which play roles in the protein functions. The peptide-micelle complex structures, including the tryptophan accessibility and dynamics were compared to circular dichroism and fluorescence studies obtained in water, trifluoroethanol and with micelles. Our work provides additional results not directly accessible by experiments that give further support to the fact that these peptides adopt an interfacial conformation within the micelles. We also show that the peptides are more buried in DDM than in DPC, and consequently, that they have a larger surface exposure to water in DPC than in DDM. As noted previously by simulations and experiments we have also observed formation of cation-π bonds between the phosphocholine DPC headgroup and Trp peptide residue. Concerning the peptide secondary structures (SS), we find that in TFE their initial helical conformations are maintained during the simulation, whereas in water their initial SS are lost after few nanoseconds of simulation. An intermediate situation is observed with micelles, where the peptides remain partially folded and more structured in DDM than in DPC. Finally, our results show no sign of ß-strand structure formation as invoked by far-UV CD experiments even when three identical peptides are simulated either in water or with micelles.
Subject(s)
Glucosides/chemistry , Molecular Dynamics Simulation , Multidrug Resistance-Associated Proteins/chemistry , Peptides/chemistry , Phosphorylcholine/analogs & derivatives , Amino Acid Sequence , Circular Dichroism , Humans , Micelles , Molecular Sequence Data , Phosphorylcholine/chemistry , Protein Binding , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multimutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick base pairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA:PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA:PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition.
Subject(s)
Hypoxanthine/chemistry , Peptide Nucleic Acids/chemistry , Proto-Oncogene Proteins/genetics , RNA, Messenger/chemistry , ras Proteins/genetics , Adenine/chemistry , Algorithms , Base Pairing , Circular Dichroism , Guanine/chemistry , Humans , Hypoxanthine/chemical synthesis , Models, Molecular , Molecular Dynamics Simulation , Nucleic Acid Denaturation , Peptide Nucleic Acids/chemical synthesis , Point Mutation , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins p21(ras) , Transition Temperature , Uracil/chemistry , ras Proteins/chemistryABSTRACT
Iron chelators, through their capacity to modulate the iron concentration in cells, are promising molecules for cancer chemotherapy. Chelators with high lipophilicity easily enter into cells and deplete the iron intracellular pool. Consequently, iron-dependent enzymes, such as ribonucleotide reductase, which is over-expressed in cancer cells, become nonfunctional. A series of calix[4]arene derivatives substituted at the lower rim by ICL670, a strong FeIII chelator, have been synthesized. Physicochemical properties and antiproliferative, angiogenesis, and tumorigenesis effects of two calix[4]arenes mono- (5a) or disubstituted (5b) with ICL670 have been studied. These compounds form metal complexes in a ratio of one to two ligands per FeIII atom as shown by combined analyses of the protometric titration curves and ESIMS spectra. The grafting of an ICL670 group on a calix[4]arene core does not significantly alter the acid-base properties, but improves the iron-chelating and lipophilicity properties. The best antiproliferative and anti-angiogenic results were obtained with calix[4]arene ligand 5a, which possesses the highest corresponding properties. Analyses of molecular dynamics simulations performed on the two calix[4]arenes provide three-dimensional structures of the complexes and proved 5a to be the most stable upon complexation.
ABSTRACT
We present the first comparative molecular dynamics investigation for a dodecylphosphocholine (DPC) micelle performed in condensed phase using the CHARMM36, GROMOS53A6, GROMOS54A7, and GROMOS53A6/Berger force fields and a set of parameters developed anew. Our potential consists of newly derived RESP atomic charges, which are associated with the Amber99SB force field developed for proteins. This new potential is expressly designed for simulations of peptides and transmembrane proteins in a micellar environment. To validate this new ensemble, molecular dynamics simulations of a DPC micelle composed of 54 monomers were carried out in explicit water using a "self-assembling" approach. Characteristic micellar properties such as aggregation kinetic, volume, size, shape, surface area, internal structure, surfactant conformation, and hydration were thoroughly examined and compared with experiments. Derived RESP charge values combined with parameters taken from Amber99SB reproduce reasonably well important structural properties and experimental data compared to the other tested force fields. However, the headgroup and alkyl chain conformations or the micelle hydration simulated with the Amber99SB force field display some differences. In particular, we show that Amber99SB slightly overestimates the trans population of the alkyl Csp(3)-Csp(3)-Csp(3)-Csp(3) dihedral angle (i.e., CCCC) and reduces the flexibility of the DPC alkyl chain. In agreement with experiments and previously published studies, the DPC micelle shows a slightly ellipsoidal shape with a radius of gyration of â¼17 Å for the different potentials evaluated. The surface of contact between the DPC headgroup and water molecules represents between 70% and 80% of the total micelle surface independently of the force field considered. Finally, molecular dynamics simulations show that water molecules form various hydrogen-bond patterns with the surfactant headgroup, as noted previously for phospholipids with a phosphatidylcholine headgroup.
ABSTRACT
Molecular dynamics simulations describing the solvation process of native and modified cyclodextrins (per-substituted α-, ß-, and γ-cyclodextrins, as well as an amino-acid derived ß-cyclodextrin) have been performed. A homogeneous force field, namely "q4md-CD", has been built from the development of a new force field topology database and from a combination of the GLYCAM04 and Amber99SB force fields to correctly describe the geometrical, structural, dynamical and hydrogen bonding aspects of heterogeneous cyclodextrin based systems. These include native, organo- and peptidic-linked cyclodextrins. q4md-CD features: (i) geometrical parameters from Amber99SB to describe the protein parts, (ii) geometrical parameters from GLYCAM04 for the carbohydrate and organic parts when available or those of Amber99SB otherwise, (iii) partial atomic charges, embedded in force field libraries for the carbohydrate and organic fragments, were derived using the R.E.D. tools according to the "Amber" strategy and (iv) scaling factors of 1.2 and 2.0 were imposed for the 1-4 electrostatic and 1-4 van der Waals interactions, respectively. Results given by q4md-CD on native cyclodextrins have been compared to those obtained with reference to force fields like GLYCAM04, GLYCAM06 and Amber99SB as well as with experimental data. This work not only gives a global view of the performances of the aforementioned force fields towards a correct description of solvated cyclodextrins, but also extends the capabilities of current force fields by addressing some issues concerning hydrogen bonding and opens new possibilities towards studies of glycoconjugates by molecular dynamics.
Subject(s)
Cyclodextrins/chemistry , Molecular Dynamics Simulation , Hydrogen Bonding , Molecular Structure , StereoisomerismABSTRACT
R.E.D. Server is a unique, open web service, designed to derive non-polarizable RESP and ESP charges and to build force field libraries for new molecules/molecular fragments. It provides to computational biologists the means to derive rigorously molecular electrostatic potential-based charges embedded in force field libraries that are ready to be used in force field development, charge validation and molecular dynamics simulations. R.E.D. Server interfaces quantum mechanics programs, the RESP program and the latest version of the R.E.D. tools. A two step approach has been developed. The first one consists of preparing P2N file(s) to rigorously define key elements such as atom names, topology and chemical equivalencing needed when building a force field library. Then, P2N files are used to derive RESP or ESP charges embedded in force field libraries in the Tripos mol2 format. In complex cases an entire set of force field libraries or force field topology database is generated. Other features developed in R.E.D. Server include help services, a demonstration, tutorials, frequently asked questions, Jmol-based tools useful to construct PDB input files and parse R.E.D. Server outputs as well as a graphical queuing system allowing any user to check the status of R.E.D. Server jobs.
Subject(s)
Software , Static Electricity , Calixarenes/chemistry , Internet , Molecular Dynamics Simulation , Quantum Theory , User-Computer InterfaceABSTRACT
This paper deals with the development and validation of new potential parameter sets, based on the CHARMM36 and GLYCAM06 force fields, to simulate micelles of the two anomeric forms (α and ß) of N-dodecyl-ß-maltoside (C(12)G(2)), a surfactant widely used in the extraction and purification of membrane proteins. In this context, properties such as size, shape, internal structure, and hydration of the C(12)G(2) anomer micelles were thoroughly investigated by molecular dynamics simulations and the results compared with experiments. Additional simulations were also performed with the older CHARMM22 force field for carbohydrates (Kuttel, M.; et al. J. Comput. Chem. 2002, 23, 1236-1243). We find that our CHARMM and GLYCAM parameter sets yield similar results in the case of properties related to the micelle structure but differ for other properties such as the headgroup conformation or the micelle hydration. In agreement with experiments, our results show that for all model potentials the ß-C(12)G(2) micelles have a more pronounced ellipsoidal shape than those containing α anomers. The computed radius of gyration is 20.2 and 25.4 Å for the α- and ß-anomer micelles, respectively. Finally, we show that depending on the potential the water translational diffusion of the interfacial water is 7-11.5 times slower than that of bulk water due to the entrapment of the water in the micelle crevices. This retardation is independent of the headgroup in α- or ß-anomers.
Subject(s)
Detergents/chemistry , Glucosides/chemistry , Micelles , Molecular Conformation , Water/chemistry , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , SoftwareABSTRACT
Multimeric lactosides based on carbohydrate scaffolds with valencies ranging from 1 to 4 and different linker lengths were synthesized by a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The binding affinities and crosslinking abilities of the new "click clusters" toward biologically relevant galectins (gal-1, gal-3) and peanut lectin were evaluated by fluorescent polarization assay (FPA) and enzyme-linked lectin assay (ELLA), respectively. FPA indicated that the binding affinities of the synthetic multilactosides towards the galectins increased proportionally with their lactosyl content, without significant differences due to the spacer length. ELLA evidenced a modest cluster effect for the multivalent conjugates, with a relative potency per lactoside ranging from 2.1 to 3.2. Nearly identical binding affinities were recorded for derivatives differing in the length of the linkers, in agreement with the FPA data. These results demonstrate that this parameter does not significantly influence the recognition process when interactions occur at a single lectin site. Molecular dynamics revealed that glycoconjugates adopt a pseudoglobular structure with a random localization of the lactoside residues. These spatial distributions were observed irrespective of the linker length; this explains the virtually equal affinities recorded by ELLA. In contrast, two-site "sandwich" ELLA clearly revealed that multivalent derivatives bearing the longest spacers were more efficient for crosslinking lectins. Intrinsic affinities, devoid of aggregation effects, and crosslinking capabilities are, therefore, not directly related phenomena that must be taking into consideration in neoglycoconjugate design for specific applications.
Subject(s)
Galectin 1/chemistry , Galectin 3/chemistry , Glycosides/chemistry , Peanut Agglutinin/chemistry , Alkynes/chemistry , Arachis/metabolism , Azides/chemistry , Catalysis , Copper/chemistry , Enzyme Assays , Fluorescence Polarization Immunoassay , Molecular Dynamics SimulationABSTRACT
Deriving atomic charges and building a force field library for a new molecule are key steps when developing a force field required for conducting structural and energy-based analysis using molecular mechanics. Derivation of popular RESP charges for a set of residues is a complex and error prone procedure because it depends on numerous input parameters. To overcome these problems, the R.E.D. Tools (RESP and ESP charge Derive, ) have been developed to perform charge derivation in an automatic and straightforward way. The R.E.D. program handles chemical elements up to bromine in the periodic table. It interfaces different quantum mechanical programs employed for geometry optimization and computing molecular electrostatic potential(s), and performs charge fitting using the RESP program. By defining tight optimization criteria and by controlling the molecular orientation of each optimized geometry, charge values are reproduced at any computer platform with an accuracy of 0.0001 e. The charges can be fitted using multiple conformations, making them suitable for molecular dynamics simulations. R.E.D. allows also for defining charge constraints during multiple molecule charge fitting, which are used to derive charges for molecular fragments. Finally, R.E.D. incorporates charges into a force field library, readily usable in molecular dynamics computer packages. For complex cases, such as a set of homologous molecules belonging to a common family, an entire force field topology database is generated. Currently, the atomic charges and force field libraries have been developed for more than fifty model systems and stored in the RESP ESP charge DDataBase. Selected results related to non-polarizable charge models are presented and discussed.
Subject(s)
Software , Algorithms , Databases, Factual , Ethanol/chemistry , Molecular Dynamics Simulation , Quantum Theory , Static Electricity , ThermodynamicsABSTRACT
The focus here is on incorporating electronic polarization into classical molecular mechanical force fields used for macromolecular simulations. First, we briefly examine currently used molecular mechanical force fields and the current status of intermolecular forces as viewed by quantum mechanical approaches. Next, we demonstrate how some components of quantum mechanical energy are effectively incorporated into classical molecular mechanical force fields. Finally, we assess the modeling methods of one such energy component-polarization energy-and present an overview of polarizable force fields and their current applications. Incorporating polarization effects into current force fields paves the way to developing potentially more accurate, though more complex, parameterizations that can be used for more realistic molecular simulations.
ABSTRACT
The web-based RESP ESP charge DataBase (R.E.DD.B., http://q4md-forcefieldtools.org/REDDB) is a free and new source of RESP and ESP atomic charge values and force field libraries for model systems and/or small molecules. R.E.DD.B. stores highly effective and reproducible charge values and molecular structures in the Tripos mol2 file format, information about the charge derivation procedure, scripts to integrate the charges and molecular topology in the most common molecular dynamics packages. Moreover, R.E.DD.B. allows users to freely store and distribute RESP or ESP charges and force field libraries to the scientific community, via a web interface. The first version of R.E.DD.B., released in January 2006, contains force field libraries for molecules as well as molecular fragments for standard residues and their analogs (amino acids, monosaccharides, nucleotides and ligands), hence covering a vast area of relevant biological applications.
Subject(s)
Databases, Factual , Static Electricity , Computational Biology , Internet , Molecular Structure , User-Computer InterfaceABSTRACT
Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5'-CCA AAG6 XA8C CGG G-3', where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites. Biochemistry, 46, 3096-3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3' to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.
Subject(s)
DNA Damage , Oligodeoxyribonucleotides/chemistry , Adenine/chemistry , Base Pairing , Base Sequence , Carbohydrate Conformation , Computer Simulation , Cytosine/chemistry , DNA/chemistry , Deoxyribose/chemistry , Guanine/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphates/chemistry , Protons , Thymine/chemistryABSTRACT
Most adults affected with HFE hereditary hemochromatosis (HH type 1, MIMmusical sharp 235200) are homozygous for the p.Cys282Tyr mutation in HFE (NC_000006.10, region 26195427 to 26205038). The aim of this study was to investigate the molecular basis of iron overload in a patient presenting with severe clinical HH with one c.845G>A (p.Cys282Tyr) allele only. Molecular and pedigree studies demonstrated the presence of the c.845G>A (p.Cys282Tyr) mutation in one allele whereas the other carried the c.187C>G (p.His63Asp) mutation plus a new c.128G>A (p.Gly43Asp) substitution in cis. A molecular modeling study of the p.[Gly43Asp;His63Asp] and p.His63Asp variants versus the wild type was carried out using molecular dynamics (MD) simulation in presence of implicit solvent. We found that the c.187C>G (p.His63Asp) mutation does not introduce any major change in the 1- domains of HFE whereas the c.128G>A (p.Gly43Asp) substitution is responsible for a modification of the dynamics and the structure of the Gln40-Ser45 loop, a critical region for HFE-TfR1 interaction thus impairing HFE-TfR1 normal contact. We conclude that the occurrence of complex alleles may be an alternative explanation for the variability of the phenotype in individuals who are compound heterozygous for c.[187C>G]+[845G>A] (p.[His63Asp]+[Cys282Tyr]).
Subject(s)
Amino Acid Substitution , Antigens, CD/metabolism , Hemochromatosis/genetics , Heterozygote , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Receptors, Transferrin/metabolism , Alleles , Amino Acids/genetics , Binding Sites , Hemochromatosis/metabolism , Hemochromatosis Protein , Histocompatibility Antigens Class I/metabolism , Humans , Male , Membrane Proteins/metabolism , Middle Aged , Models, Molecular , Mutation, Missense , Pedigree , Structure-Activity RelationshipABSTRACT
A short and efficient strategy for the synthesis of multi-valent mannosides based on a selectively functionalized carbohydrate scaffold was reported involving (i) direct regioselective azidation of unprotected commercial saccharides, (ii) acetylation, (iii) grafting of the mannosyl ligands by click chemistry, and (iv) deacetylation. New glycoclusters with a valency ranging from 1 to 4 and different spatial arrangements of the epitopes were obtained. Binding affinities of the new glycoclusters toward concanavalin A (Con A) lectin were investigated by an enzyme-linked lectin essay (ELLA). The synthetic multi-valent compounds exhibited a remarkable cluster effect with a relative potency per mannoside residue ranging from 8.1 to 9.1 depending on the structures. ELLA experiments were in agreement with the establishment of favorable interactions between triazole ring and Con A, increasing the binding affinity. A new force field topology database was developed in agreement with the GLYCAM 2004 force field. Molecular dynamics performed on representative glyco-conjugates revealed interesting structural features such as rigidity of the scaffold for a well-defined presentation of the ligands and highly flexible mannose counterparts. The new glycoconjugates reported may be promising tools as probes or effectors of biological processes involving lectins.
Subject(s)
Concanavalin A/chemical synthesis , Lectins/chemical synthesis , Mannosides/chemistry , Acetylation , Azides/chemistry , Binding Sites , Carbohydrate Conformation , Carbohydrate Sequence , Concanavalin A/metabolism , Lectins/metabolism , Ligands , Mannosides/metabolism , Models, Chemical , Molecular Sequence Data , Triazoles/chemistryABSTRACT
A 4'-oxidized abasic site (X) has been synthesized in a defined duplex DNA sequence, 5'-d(CCAAAGXACCGGG)-3'/3'-d(GGTTTCATGGCCC)-5' (1). Its structure has been determined by two-dimensional NMR methods, molecular modeling, and molecular dynamics simulations. 1 is globally B-form with the base (A) opposite X intrahelical and well-stacked. Only the alpha anomer of X is observed, and the abasic site deoxyribose is largely intrahelical. These results are compared with a normal abasic site (Y) in the same sequence context (2). Y is composed of a 60:40 mixture of alpha and beta anomers (2alpha and 2beta). In both 2alpha and 2beta, the base (A) opposite Y is intrahelical and well-stacked and the abasic site deoxyribose is predominantly extrahelical, consistent with the reported structures of the normal abasic site in a similar sequence context [Hoehn, S. T., Turner, C. J., and Stubbe, J. (2001) Nucleic Acids Res. 29, 3413-3423]. Molecular dynamics simulations reveal that the normal abasic site appears to be conformationally more flexible than the 4'-oxidized abasic site. The importance of the structure and flexibility of the abasic site in the recognition by the DNA repair enzyme Ape1 is discussed.
Subject(s)
DNA Damage/drug effects , DNA/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Electron Spin Resonance Spectroscopy , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Oxidation-ReductionABSTRACT
2-(2'-Hydroxyphenyl)benzoxazole (HBO) may be used as a model base pair to study solvation, duplex environment, and tautomerization within the major and minor groves of DNA duplexes. In its ground state, HBO possesses an enol moiety which may be oriented syn or anti relative to the imino nitrogen of the benzoxazole ring. In the absence of external hydrogen-bond donors and acceptors HBO exists as the internally hydrogen-bonded syn-enol, a mimic of the rare base pair tautomer found in DNA, which may be photoinduced to tautomerize and form the keto tautomer, a mimic of the dominant base pair tautomer. Previously, we demonstrated that when incorporated into DNA such that the enol moiety is positioned in the major groove, HBO is not solvated, exists exclusively as the internally hydrogen-bonded syn-enol which is efficiently photoinduced to tautomerize, and the corresponding keto tautomer is preferentially stabilized. In stark contrast, we now show that when HBO is incorporated in DNA such that the enol moiety is positioned in the minor groove, the enol tautomer is preferentially stabilized. Molecular dynamics simulations suggest that this results from the formation of a stable hydrogen-bond between the HBO enol and the O4' atom of an adjacent nucleotide, an H-bond acceptor that is only available in the minor groove. The differential stabilization of the enol and keto tautomers in the major and minor grooves may reflect the functions for which these environments evolved, including duplex replication, stability, and recognition.
Subject(s)
Base Pairing , Benzoxazoles/chemistry , DNA/chemistry , DNA/ultrastructure , Isomerism , Models, Molecular , Nucleic Acid Conformation , Protons , SolubilityABSTRACT
In Caucasians, 4-35% of hemochromatosis patients carry at least one chromosome without a common HFE mutation (i.e. C282Y, H63D and S65C). Several studies have now shown that iron overload phenotypes in such patients can be associated with uncommon HFE mutations. We previously supported implication of the C282Y/Q283P compound heterozygous genotype in hemochromatosis phenotypes and, based on molecular dynamics simulations, proposed that the Q283P substitution prevents normal folding of the HFE alpha3-domain. In the current work, we have used HeLa cells carrying wild-type or Q283P-mutant HFE cDNA under the control of a tetracycline-sensitive promoter to functionally characterise the Q283P mutation. Experiments using cells over-expressing wild-type HFE confirm the existence of beta2microglobulin(beta2m)/HFE and HFE/transferrin receptor 1 (TfR1) interactions, as well as the capacity of HFE to reduce transferrin-mediated iron uptake. In contrast, neither beta2m/HFE nor HFE/TfR1 complex formation was detected in cells over-expressing the mutated form of HFE. Moreover, the 283P HFE protein was found to have a very limited effect on the major cellular iron uptake pathway. Combined, our results indicate that the Q283P mutation leads to structural and functional consequences similar to those described for the main hereditary hemochromatosis mutation. As a consequence, our study has implications for the screening of hemochromatosis patients that have one or two copies of HFE which lack the main mutations. It also highlights that protein structure prediction methods could be more generally used to better interpret relationships between rare genotypes and molecular diagnosis of a human inherited disorder.
Subject(s)
Hemochromatosis/genetics , Histocompatibility Antigens Class I/genetics , Membrane Proteins/genetics , Mutation, Missense , Antigens, CD/metabolism , DNA, Complementary , HeLa Cells , Hemochromatosis/metabolism , Hemochromatosis Protein , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Humans , Iron/pharmacokinetics , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Polymerase Chain Reaction , Receptors, Transferrin/metabolism , TransfectionABSTRACT
Far-UV photolysis of 4-thiothymidylyl(3'-5')thymidine led to the formation of three stable derivatives: one resulting from a combination between a 3'-end methylene radical and a 5'-end C(4) radical [4-(alpha-thyminyl) derivative] and two formed after a combination between a 3'-end methylene radical and a 5'-end C(6) radical [6-(alpha-thyminyl) derivative]. In the latter series, two stereochemical pathways took place during the reaction between the methylene and C(6) radicals. The major pathway occurred when the 5'-base glycosidic bond had an anti conformation leading to an S configuration of the C(6) Tp-end. The minor pathway, which had never been reported before in this series, involved a 5'-base in a syn conformation leading consequently to the R configuration at the C(6) Tp-end. The 5,6-dihydrothymine moiety of these two adducts presented a 5,6-trans diaxial substitution that resulted from the epimerization, at the 5,6-dihydropyrimidine 5-position, of a less stable cis-disubstituted intermediate.
Subject(s)
DNA/radiation effects , Thymine/analogs & derivatives , Thymine/chemical synthesis , Gamma Rays , Molecular Structure , Photolysis , Pyrimidines/chemistry , StereoisomerismABSTRACT
The increase in computer power and the development of new mathematical concepts implemented in software have allowed computational chemistry to emerge as a new research field. Although programs were freely distributed during the "golden age" of this discipline, today they are usually copyrighted and have become easier and easier to use through sophisticated graphical interfaces. This "democratization" is a vector of success for this discipline. Nowadays, non-theoreticians can use such programs more easily and solve chemistry-related problems with the computer. The number of program offerings has rapidly grown and private companies specialized in molecular modeling have appeared and compete to sell their products. Thus, numerous software packages, often presenting similar capabilities, are now available on the market. Within this context, the availability of the program source code remains, in our opinion, an important criterion for program selection.