Processes of base-pair opening and proton exchange in Z-DNA

Using proton magnetic resonance, we have investigated imino and amino proton exchange in the Z form of the four oligomers d(Cbr8GCGCbr8G), d(CGm5CGCG), d(CG)6, and d(CG)12. In the latter two oligomers, all five exchangeable protons have been assigned. We find that proton acceptors such as NH3 or the basic form of Tris enhance imino proton exchange. The base-pair lifetime can then be obtained by extrapolation of the exchange time to infinite concentration of proton acceptor. For d(CG)6 and d(CG)12, the values are ca. 3.5 ms at 80 degrees C and ca. 130 ms at 35 degrees C. The latter value is about 65 times longer than in the same oligomers in the B form. The activation energy of base-pair opening, 80 kJ/mol, is the same in the Z and the B forms of d(CG)12. At 5 degrees C, the base-pair lifetime is about 3 s, much smaller than the time constant of the Z to B transition, to which it is therefore unrelated. The base-pair dissociation constant at 35 degrees C, 0.5 X 10(-6), is 5 times smaller than for the same oligomers in the B form. In the absence of added catalyst, at pH 7, the exchange time of the imino proton is 30 min at 5 degrees C. That of both cytidine amino protons, assigned by NOE, is about 50 min. The longest proton exchange time, ca. 330 min, is assigned unambiguously to the guanosine amino protons. Thus assigned and interpreted in terms of exchange chemistry rather than structural kinetics, the exchange times do not support earlier models of Z-DNA internal motions.     

Crystal structure of a Z-DNA fragment containing thymine/2-aminoadenine base pairs

The Z-DNA structure has been shown to form in two crystals made from self-complementary DNA hexamers d(CGTDCG) and d(CDCGTG) which contain thymine/2-aminoadenine (TD) base pairs. The latter structure has been solved and refined to 1.3 A resolution and it shows only small conformational changes due to the introduction of the TD base pairs in comparison with the structure of d(CG)3. Spectroscopic studies with these compounds demonstrate that DNA molecules containing 2-aminoadenine residues form Z-DNA slightly more easily than do those containing adenine nucleotides, but not as readily as the parent sequence containing only guanine-cytosine base pairs.     

Structure of d(CACGTG), a Z-DNA hexamer containing AT base pairs.

The left-handed Z-DNA conformation has been observed in crystals made from the self-complementary DNA hexamer d(CACGTG). This is the first time that a non disordered Z form is found in the crystal structure of an alternating sequence containing AT base pairs without methylated or brominated cytosines. The structure has been determined and refined to an agreement factor R = 22.9% using 746 reflections in the resolution in the resolution shell 7 to 2.5 A. The overall shape of the molecule is very similar to the Z-structure of the related hexamer d(CG)3 confirming the rigidity of the Z form. No solvent molecules were detected in the minor groove of the helix near the A bases. The disruption of the spine of hydration in the AT step appears to be a general fact in the Z form in contrast with the B form. The biological relevance of the structure in relation to the CA genome repeats is discussed.     

The intrinsic structure and stability of out-of-alternation base pairs in Z-DNA.

Alternating pyrimidine-purine sequences typically form Z-DNA, with the pyrimidines in the anti and purines in the syn conformations. The observation that dC and dT nucleotides can also adopt the syn conformation (i.e. the nucleotides are out-of-alternation) extends the range of sequences that can convert to this left-handed form of DNA. Here, we study the effects of placing two adjacent d(G*C) base pairs as opposed to a single d(G*C) base pair or two d(A*T) base pairs out-of-alternation by comparing the structure of d(m5CGGCm5CG)2with the previously published structures of d(m5CGGGm5CG)*d(m5CGCCm5CG) and d(m5CGATm5CG)2. A high buckle and loss of stacking interactions are observed as intrinsic properties of the out-of-alternation base pairs regardless of sequence and the context of the dinucleotide. From solution titrations, we find that the destabilizing effect of out-of-alternation d(G*C) base pairs are identical whether these base pairs are adjacent or isolated. We can therefore conclude that it is these intrinsic distortions in the structure of the base pairs and not neighboring effects that account for the inability of out-of-alternation base pairs to adopt the left-handed Z conformation.     

High base pair opening rates in tracts of GC base pairs

Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.     

AT base pairs are less stable than GC base pairs in Z-DNA: The crystal structure of d(m5CGTAm5CG)

Two hexanucleoside pentaphosphates, 5-methyl and 5-bromo cytosine derivatives of d(CpGpTp-ApCpG) have been synthesized, crystallized, and their three-dimensional structure solved. They both form left-handed Z-DNA and the methylated derivative has been refined to 1.2 Å resolution. These are the first crystal Z-DNA structures that contain AT base pairs. The overall form of the molecule is very similar to that of the unmethylated or the fully methylated (dC-dG)₃ hexamer although there are slight changes in base stacking. However, significant differences are found in the hydration of the helical groove. When GC base pairs are present, the helical groove is systematically filled with two water molecules per base pair hydrogen bonded to the bases. Both of these water molecules are not seen in the electron density map in the segments of the helix containing AT base pairs, probably because of solvent disorder. This could be one of the features that makes AT base pairs form Z-DNA less readily than GC base pairs.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

Protein hydrogen exchange mechanism: local fluctuations

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability. The pattern of HX changes shows that the coupled structural distortions that dominate exchange can be several residues in extent, but they expose to exchange only one amide NH at a time. This "local fluctuation" mode of hydrogen exchange may be generally recognized by disparate near-neighbor rates and a low dependence on destabilants (denaturant, temperature, pressure). In contrast, concerted unfolding reactions expose multiple neighboring amide NHs with very similar computed protection factors, and they show marked destabilant sensitivity. In both modes, ionic hydrogen exchange catalysts attack from the bulk solvent without diffusing through the protein matrix.     

Phosphatidylethanolamine synthesis by castor bean endosperm : a base exchange reaction

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl(2). l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate.     

Probing the nature of hydrogen bonds in DNA base pairs

Energy decomposition analyses based on the block-localized wave-function (BLW-ED) method are conducted to explore the nature of the hydrogen bonds in DNA base pairs in terms of deformation, Heitler–London, polarization, electron-transfer and dispersion-energy terms, where the Heitler–London energy term is composed of electrostatic and Pauli-exchange interactions. A modest electron-transfer effect is found in the Watson–Crick adenine–thymine (AT), guanine–cytosine (GC) and Hoogsteen adenine-thymine (H-AT) pairs, confirming the weak covalence in the hydrogen bonds. The electrostatic attraction and polarization effects account for most of the binding energies, particularly in the GC pair. Both theoretical and experimental data show that the GC pair has a binding energy (−25.4 kcal mol⁻¹ at the MP2/6-31G** level) twice that of the AT (−12.4 kcal mol⁻¹) and H-AT (−12.8 kcal mol⁻¹) pairs, compared with three conventional N-H···O(N) hydrogen bonds in the GC pair and two in the AT or H-AT pair. Although the remarkably strong binding between the guanine and cytosine bases benefits from the opposite orientations of the dipole moments in these two bases assisted by the π-electron delocalization from the amine groups to the carbonyl groups, model calculations demonstrate that π-resonance has very limited influence on the covalence of the hydrogen bonds. Thus, the often adopted terminology “resonance-assisted hydrogen bonding (RHAB)” may be replaced with “resonance-assisted binding” which highlights the electrostatic rather than electron-transfer nature of the enhanced stabilization, as hydrogen bonds are usually regarded as weak covalent bonds. Figure Electron density difference (EDD) maps for the GC pair: a shows the polarization effect (isodensity 1.2×10⁻³ a.u.); b shows the charge transfer effect (isodensity 2×10⁻⁴ a.u.) Dedicated to Professor Paul von Ragué Schleyer on the occasion of his 75th birthday     

Effects of 5-fluorouracil/guanine wobble base pairs in Z-DNA: molecular and crystal structure of d(CGCGFG).

The chemotherapeutic agent 5-fluorouracil is a DNA base analogue which is known to incorporate into DNA in vivo. We have solved the structure of the oligonucleotide d(CGCGFG), where F is 5-fluorouracil (5FU). The DNA hexamer crystallizes in the Z-DNA conformation at two pH values with the 5FU forming a wobble base pair with guanine in both crystal forms. No evidence of the enol or ionized form of 5FU is found under either condition. The crystals diffracted X-rays to a resolution of 1.5 A and their structures have been refined to R-factors of 20.0% and 17.2%, respectively, for the pH = 7.0 and pH = 9.0 forms. By comparing this structure to that of d(CGCGCG) and d(CGCGTG), we were able to demonstrate that the backbone conformation of d(CGCGFG) is similar to that of the archetypal Z-DNA. The two F-G wobble base pairs in the duplex are structurally similar to the T-G base pairs both with respect to the DNA helix itself and its interactions with solvent molecules. In both cases water molecules associated with the wobble base pairs bridge between the bases and stabilize the structure. The fluorine in the 5FU base is hydrophobic and is not hydrogen bonded to any solvent molecules.     

Vibrational fluctuations of hydrogen bonds in a DNA double helix with nonuniform base pairs.

The Green's function technique is applied to a study of breathing modes in a DNA double helix which contains a region of different base pairs from the rest of the double helix. The calculation is performed on a G-C helix in the B conformation with four consecutive base pairs replaced by A-T. The average stretch in hydrogen bonds is found amplified around the A-T base pair region compared with that of poly(dG)-poly(dC). This is likely related to the A-T regions lower stability against hydrogen bond melting. The A-T region may be considered to be the initiation site for melting in such a helix.     

Loss of G-A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif

Upon binding to the 15.5K protein, two tandem-sheared G–A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA (‘k–e motion’) were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k–e motion in Kt-U4 is triggered both by loss of G–A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G–A base pairs is correlated along the first mode but anti-correlated along the third mode with the k–e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G–A base pairs and modified stacking interactions. Thus, loss of G–A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G–A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems.     

NMR spectroscopic study of a DNA duplex with mercury-mediated T-T base pairs

Recently, we reported that T-T mismatches can specifically recognize Hg(II) (T-Hg(II)-T pair formation). In order to understand the properties of the T-Hg(II)-T pair, we recorded NMR spectra for a DNA duplex, d(CGCGTTGTCC).d(GGACTTCGCG), with two successive T-T mismatches (Hg (II)-binding sites). We assigned 1H resonances for mercury-free and di-mercurated duplexes, and performed titration experiments with Hg(II) by using 1D 1H NMR spectra. Because of the above mentioned assignments, we could confirm the existence of mono-mercurated species, because individual components gave independent NMR signals in the titration spectra.     

基于密度泛函理论研究Watson–Crick碱基对中的氢键特征(英文)

基于密度泛函理论(DFT)研究腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶以及腺嘌呤胸腺嘧啶碱基对、鸟嘌呤胞嘧啶碱基对。在DFT-B3LYP/6-31G**水平上利用自然键轨道理论分析研究结果显示,互补碱基对的结构和电子特征有利于氢键的形成。本文中讨论几何结构、电子结构、分子轨道和能量对于氢键形成的影响。此研究结果将有助于更好的理解AT和GC碱基对中氢键与它们的结构特性之间的关系。     

Evidence for a bound water molecule next to the retinal Schiff base in bacteriorhodopsin and rhodopsin: a resonance Raman study of the Schiff base hydrogen/deuterium exchange.

The retinal chromophores of both rhodopsin and bacteriorhodopsin are bound to their apoproteins via a protonated Schiff base. We have employed continuous-flow resonance Raman experiments on both pigments to determine that the exchange of a deuteron on the Schiff base with a proton is very fast, with half-times of 6.9 +/- 0.9 and 1.3 +/- 0.3 ms for rhodopsin and bacteriorhodopsin, respectively. When these results are analyzed using standard hydrogen-deuteron exchange mechanisms, i.e., acid-, base-, or water-catalyzed schemes, it is found that none of these can explain the experimental results. Because the exchange rates are found to be independent of pH, the deuterium-hydrogen exchange can not be hydroxyl (or acid-)-catalyzed. Moreover, the deuterium-hydrogen exchange of the retinal Schiff base cannot be catalyzed by water acting as a base because in that case the estimated exchange rate is predicted to be orders of magnitude slower than that observed. The relatively slow calculated exchange rates are essentially due to the high pKa values of the Schiff base in both rhodopsin (pKa > 17) and bacteriorhodopsin (pKa approximately 13.5). We have also measured the deuterium-hydrogen exchange of a protonated Schiff base model compound in aqueous solution. Its exchange characteristics, in contrast to the Schiff bases of the pigments, is pH-dependent and consistent with the standard base-catalyzed schemes. Remarkably, the water-catalyzed exchange, which has a half-time of 16 +/- 2 ms and which dominates at pH 3.0 and below, is slower than the exchange rate of the Schiff base in rhodopsin and bacteriorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of base substituents on the hydration of B- and Z-DNA: correlations to the B- to Z-DNA transition.

We present a study of how substituent groups of naturally occurring and modified nucleotide bases affect the degree of hydration of right-handed B-DNA and left-handed Z-DNA. A comparison of poly(dG-dC) and poly(dG-dm5C) titrations with the lipotropic salts of the Hofmeister series infers that the methyl stabilization of cytosines as Z-DNA is primarily a hydrophobic effect. The hydration free energies of various alternating pyrimidine-purine sequences in the two DNA conformations were calculated as solvent free energies from solvent accessible surfaces. Our analysis focused on the N2 amino group of purine bases that sits in the minor groove of the double helix. Removing this amino group from guanine to form inosine (I) destabilizes Z-DNA, while adding this group to adenines to form 2-aminoadenine (A') stabilizes Z-DNA. These predictions were tested by comparing the salt concentrations required to crystallize hexanucleotide sequences that incorporate d(CG), d(CI), d(TA) and d(TA') base pairs as Z-DNA. Combining the current results with our previous analysis of major groove substituents, we derived a thermodynamic cycle that relates the systematic addition, deletion, or substitution of each base substituent to the B- to Z-DNA transition free energy.     

Study of the hydrogen bond in different orientations of adenine-thymine base pairs: An <Emphasis Type="Italic">ab initio</Emphasis> study

In order to gain deeper insight into structure, charge distribution, and energies of A-T base pairs, we have performed quantum chemical ab initio and density functional calculations at the HF (Hartree-Fock) and B3LYP levels with 3-21G*, 6-31G*, 6-31G**, and 6-31++G** basis sets. The calculated donor-acceptor atom distances in the Watson-Crick A-T base pair are in good agreement with the experimental mean values obtained from an analysis of 21 high resolution DNA structures. In addition, for further correction of interaction energies between adenine and thymine, the basis set superposition error (BSSE) associated with the hydrogen bond energy has been computed via the counterpoise method using the individual bases as fragments. In the Watson-Crick A-T base pair there is a good agreement between theory and experimental results. The distances for (N2...H23-N19), (N8-H13...O24), and (C1...O18) are 2.84, 2.94, and 3.63 A, respectively, at B3LYP/6-31G** level, which is in good agreement with experimental results (2.82, 2.98, and 3.52 A). Interaction energy of the Watson-Crick A-T base pair is -13.90 and -10.24 kcal/mol at B3LYP/6-31G** and HF/6-31G** levels, respectively. The interaction energy of model (9) A-T base pair is larger than others, -18.28 and -17.26 kcal/mol, and for model (2) is the smallest value, -13.53 and -13.03 kcal/mol, at B3LYP/6-31G** and B3LYP/6-31++G** levels, respectively. The computed B3LYP/6-31G** bond enthalpies for Watson-Crick A-T pairs of -14.4 kcal/mol agree well with the experimental results of -12.1 kcal/mol deviating by as little as -2.3 kcal/mol. The BSSE of some cases is large (9.85 kcal/mol) and some is quite small (0.6 kcal/mol).