Role of hoogsteen edge hydrogen bonding at template purines in nucleotide incorporation by human DNA polymerase iota

Human DNA polymerase iota (Pol iota) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol iota with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol iota active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N(6) of an A or the O(6) of a G, here we provide evidence that whereas hydrogen bonding at N(6) is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O(6) is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O(6) and N7 hydrogen bonding to DNA synthesis by Pol iota, we have examined its proficiency for replicating through the (6)O-methyl guanine and 8-oxoguanine lesions, which affect the O(6) and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O(6) is an imperative. The dispensability of N(6) hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol iota with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N(6) positions of templating A.     

Computer graphics presentations and analysis of hydrogen bonds from molecular dynamics simulation

To obtain better insights into the dynamic nature of hydrogen bonding, computer graphics representations were introduced as an aid for the analysis of molecular dynamics trajectories. A schematic representation of hydrogen bonding patterns is generated to reflect the frequency and the type of hydrogen bonding occurring during the simulation period. Various trajectory plots for monitoring geometrical parameters and for analyzing three-center hydrogen bonding were also generated. The methods proposed are applicable to a variety of biopolymers. In this study, hydrogen bonding in the d(G) · d(C)₆ system was examined. For the nucleic acid fragments examined, three-center hydrogen bonds can be classified as in-plane and major or minor groove types. The in-plane three-center hydrogen bond represents a stable state in which both bonds simultaneously satisfy the relaxed hydrogen bonding criteria for a measurable period. On the other hand, groove three-center hydrogen bonds behave as a transient intermediate state in a flip-flop hydrogen bonding system.     

Molecular mechanics calculations on a triple stranded DNA involving C+.G-T and T.A+-C mismatched base triples

We have carried out molecular modeling of a triple stranded pyrimidine(Y). purine(R): pyrimidine(Y) (where ':' refers to Watson-Crick and '.' to Hoogsteen bonding) DNA, formed by a homopurine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC). Molecular mechanics calculations using NMR constraints have provided a detailed three dimensional structure of the triplex. The entire stretches of purine and the pyrimidine nucleotides have a conformation close to B-DNA. The three strands are held by the canonical C+.G:C and T.A:T hydrogen bonds. The structure also contains two mismatch C+.G-T and T.A+-C base triples which have been characterized for the first time. In the A+-C base-pair of the T.A+-C triple, both hydrogen donors are situated on the purine (A+(1N) and A+(6N)). We observe a unique hydrogen bonding interaction scheme in case of C+.G-T where one acceptor, G(60), is bonded to three donors (C+(3NH), C+(4NH2) and T(3NH)). Though the C+.G-T base triple is less stable than C+.G:C, it is significantly more stable than T.A:T. On the other hand, T.A+-C is as stable as the T.A:T base triad.     

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.     

Computational study of the absorption spectra of green fluorescent protein mutants

In this work, we present a theoretical study of the relationship between molecular structure and the red-shift in absorption spectra of S65G and S65T green fluorescent protein (GFP) mutants. To identify the effects of the protein environment, we combined results from molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics calculations to obtain structural properties, and applied time-dependent density functional theory to calculate the excitation energies. By using results from the MD simulations, we were able to provide a systematic analysis of the structural details that may effect the red-shift in the absorption spectra when taking into account temperature effects. Furthermore, a detailed study of hydrogen bonding during the MD simulations demonstrated differences between S65G and S65T, for example, regarding hydrogen bonding with Glu222. An analysis of the absorption spectra for different forms of the chromophore emphasized the dominance of the anionic forms in solution for the S65G and S65T GFP mutants.     

Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure

The stacking and hydrogen bonding abilities of Trp-(Gly)n-Glu (n = 0 approximately 3) for the interaction with 7-methylguanine (m7G) base were examined by fluorescence and 1H-NMR methods, and it was shown that they correlate with the distance between the Trp and Glu residues, and become most significant when both residues are separated from each other by two Gly residues (n = 2). Based on this insight, the sequence conserved between the human and yeast cap binding proteins (CBPs) was surveyed, and the sequence of Trp-Glu-Asp-Glu (No. 102-105 in human CBP) was selected as a probable site for the binding with mRNA cap structure. Thus, the stacking and hydrogen bonding abilities of Trp-Glu-Asp-Glu with m7G cap structure were examined by comparative experiments using its analogous peptides. The results showed that the fourth Glu residue is important not only for the construction of hydrogen bond pairing with m7G base but also for strengthening the stacking interaction between the Trp indole ring and m7G base. Taking account of the recognition analysis using the mutant CBP proteins by site-directed mutagenesis (Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Morioka, H., Tanaka, T., Nishikawa, S. and Uesugi, S. (1991) FEBS Lett. 280, 207-210), this cooperative interaction could be important for the recognition of mRNA cap structure.     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen bonding modulates binding of exogenous ligands in a myoglobin proximal cavity mutant.

In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. (1)H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.     

Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli

The hydrogen bonding interactions between the Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease inactivated (KF(-)) and the minor groove of DNA were examined with modified oligodeoxynucleotides in which 3-deazaguanine (3DG) replaced guanine. This substitution would prevent a hydrogen bond from forming between the polymerase and that one site on the DNA. If the hydrogen bonding interaction were important, then we should observe a decrease in the rate of reaction. The steady-state and pre-steady-state kinetics of DNA replication were measured with 10 different oligodeoxynucleotide duplexes in which 3DG was placed at different positions. The largest decrease in the rate of replication was observed when 3DG replaced guanine at the 3'-terminus of the primer. The effect of this substitution on mispair extension and formation was then probed. The G to 3DG substitution at the primer terminus decreased the k(pol) for the extension past G/C, G/A, and G/G base pairs but not the G/T base pair. The G to 3DG substitution at the primer terminus also decreased the formation of correct base pairs as well as incorrect base pairs. However, in all but two mispairs, the effect on correct base pairs was much greater than that of mispairs. These results indicate that the hydrogen bond between Arg668 and the minor groove of the primer terminus is important in the fidelity of both formation and extension of mispairs. These experiments support a mechanism in which Arg668 forms a hydrogen bonding fork between the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP to align the 3'-hydroxyl group with the alpha-phosphate of the dNTP. This is one mechanism by which the polymerase can use the geometry of the base pairs to modulate the rate of formation and extension of mispairs.     

Molecular dynamics simulations of a beta-hairpin fragment of protein G: balance between side-chain and backbone forces

How is the native structure encoded in the amino acid sequence? For the traditional backbone centric view, the dominant forces are hydrogen bonds (backbone) and phi-psi propensity. The role of hydrophobicity is non-specific. For the side-chain centric view, the dominant force of protein folding is hydrophobicity. In order to understand the balance between backbone and side-chain forces, we have studied the contributions of three components of a beta-hairpin peptide: turn, backbone hydrogen bonding and side-chain interactions, of a 16-residue fragment of protein G. The peptide folds rapidly and cooperatively to a conformation with a defined secondary structure and a packed hydrophobic cluster of aromatic side-chains. Our strategy is to observe the structural stability of the beta-hairpin under systematic perturbations of the turn region, backbone hydrogen bonds and the hydrophobic core formed by the side-chains, respectively. In our molecular dynamics simulations, the peptides are solvated. with explicit water molecules, and an all-atom force field (CFF91) is used. Starting from the original peptide (G41EWTYDDATKTFTVTE56), we carried out the following MD simulations. (1) unfolding at 350 K; (2) forcing the distance between the C(alpha) atoms of ASP47 and LYS50 to be 8 A; (3) deleting two turn residues (Ala48 and Thr49) to form a beta-sheet complex of two short peptides, GEWTYDD and KTFTVTE; (4) four hydrophobic residues (W43, Y45, F52 and T53) are replaced by a glycine residue step-by-step; and (5) most importantly, four amide hydrogen atoms (T44, D46, T53, and T55, which are crucial for backbone hydrogen bonding), are substituted by fluorine atoms. The fluorination not only makes it impossible to form attractive hydrogen bonding between the two beta-hairpin strands, but also introduces a repulsive force between the two strands due to the negative charges on the fluorine and oxygen atoms. Throughout all simulations, we observe that backbone hydrogen bonds are very sensitive to the perturbations and are easily broken. In contrast, the hydrophobic core survives most perturbations. In the decisive test of fluorination, the fluorinated peptide remains folded under our simulation conditions (5 ns, 278 K). Hydrophobic interactions keep the peptide folded, even with a repulsive force between the beta-strands. Thus, our results strongly support a side-chain centric view for protein folding.     

Structure and activity of Y-class DNA polymerase DPO4 from Sulfolobus solfataricus with templates containing the hydrophobic thymine analog 2,4-difluorotoluene

The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to probe the relative importance of shape and hydrogen bonding for correct nucleotide insertion by DNA polymerases. As far as high fidelity (A-class) polymerases are concerned, shape is considered by some as key to incorporation of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed kinetic analysis of in vitro primer extension opposite DFT-containing templates by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus. Although full-length product formation was observed, steady-state kinetic data show that dATP insertion opposite DFT is greatly inhibited relative to insertion opposite T (approximately 5,000-fold). No products were observed in the pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP opposite DFT over dGTP (approximately 200-fold) and that the polymerase is able to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4 in complex with DNA duplexes containing the DFT analog, the first for any DNA polymerase. In the structures, template-DFT is either positioned opposite primer-A or -G at the -1 site or is unopposed by a primer base and followed by a dGTP:A mismatch pair at the active site, representative of a -1 frameshift. The three structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although hydrogen bonding is clearly important for error-free replication by this Y-class DNA polymerase, our work demonstrates that Dpo4 also relies on shape and electrostatics to distinguish between correct and incorrect incoming nucleotide.     

Theoretical studies on protein-nucleic acid interactions. II. Hydrogen bonding of amino acid side chains with bases and base pairs of nucleic acids

With a view to understanding the role of hydrogen bonds in the recognition of nucleic acids by proteins, hydrogen bonding between the bases and base pairs of nucleic acids and the amino acids (Asn, Gln, Asp and Glu, and charged residues Arg⁺, Glu^?, and Asp^?) has been studied by a second-order perturbation theory. Binding energies have been calculated for all possible configurations involving a pair of hydrogen bonds between the base (or base pair) and the amino acid residue. Our results show that the hydrogen bonding in these cases has a large contribution from electrostatic interaction. In general, the charged amino acids, compared to the uncharged ones, form more stable complexes with bases or base pairs. The hydrogen-bond energies are an order of magnitude smaller than the Coulombic interaction energies between basic amino acids (Lys⁺, Arg⁺, and His⁺) and the phosphate groups of nucleic acids. The stabilities of the complexes of amino acids Asn, Gln, Asp, and Glu with bases are in the order: G–X > C–X > A–X U–X or T–X, and G · C–X > A · T(U)–X, where X is one of these amino acid residues. It has been shown that Glu^? and Asp^? can recognize guanine in single-stranded nucleic acids; Arg⁺ can recognize G · C base pairs from A · T base pairs in double-stranded structures.     

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.     

Hydrogen bonding stabilizes globular proteins.

It is clear that intramolecular hydrogen bonds are essential to the structure and stability of globular proteins. It is not clear, however, whether they make a net favorable contribution to this stability. Experimental and theoretical studies are at odds over this important question. Measurements of the change in conformational stability, delta (delta G), for the mutation of a hydrogen bonded residue to one incapable of hydrogen bonding suggest a stabilization of 1.0 kcal/mol per hydrogen bond. If the delta (delta G) values are corrected for differences in side-chain hydrophobicity and conformational entropy, then the estimated stabilization becomes 2.2 kcal/mol per hydrogen bond. These and other experimental studies discussed here are consistent and compelling: hydrogen bonding stabilizes globular proteins.     

Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme d-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317.

According to the three-dimensional structure of a porcine kidney D-amino acid oxidase-substrate (D-leucine) complex model, the G313 backbone carbonyl recognizes the substrate amino group by hydrogen bonding and the side-chain hydroxyl of T317 forms a hydrogen bond with C(2)=O of the flavin moiety of FAD [Miura et al. (1997) J. Biochem. 122, 825-833]. We have designed and expressed the G313A and T317A mutants and compared their enzymatic and spectroscopic properties with those of the wild type. The G313A mutant shows decreased activities to various D-amino acids, but the pattern of substrate specificity is different from that of the wild type. The results imply that the hydrogen bond between the G313 backbone carbonyl and the substrate amino group plays important roles in substrate recognition and in defining the substrate specificity of D-amino acid oxidase. The T317A mutant shows a decreased affinity for FAD. The steady-state kinetic measurements indicate diminished activities of T317A to substrate D-amino acids. The transient kinetic parameters measured by stopped-flow spectroscopy revealed that T317 plays key roles in stabilizing the purple intermediate, a requisite intermediate in the oxidative half-reaction, and in enhancing the release of the product from the active site, thereby optimizing the overall catalytic process of D-amino acid oxidase.     

Poly(8-aminoguanylic acid): formation of ordered self-structures and interaction with poly(cytidylic acid).

Poly(8-aminoguanylic acid) has in neutral solution a novel ordered structure of high stability. The 8-amino group permits formation of three hydrogen bonds between two residues along the "top", or long axis, of the purines. The usual hydrogen bonding protons and Watson-Crick pairing sites are not involved in the association. The bonding scheme has a twofold rotation axis and is hemiprotonated at N(7). Poly(8NH2G) is converted by alkaline titration (pK = 9.7) to a quite different ordered structure, which is the favored form over the range approximately pH 10-11. The bonding scheme appears to be composed of a planar, tetrameric array of guanine residues, in which the 8-amino group does not participate in interbase hydrogen bonding. Poly (8NH2G) does not interact with poly(C) in neutral solution because of the high stability of the hemiprotonated G-G self-structure. Titration to the alkaline plateau, however, permits ready formation of a two-stranded Watson-Crick helix. In contrast to the monomer 8NH2GMP, poly(8NH2G) does not form a triple helix with poly(C) under any conditions. The properties of the ordered structures are interpreted in terms of a strong tendency of the 8-amino group to form a third interbase hydrogen bond, when this possibility is not prevented by high pH.     

Choice of base at silent codon site 3 is not selectively neutral in eucaryotic structural genes: It maintains excess short runs of weak and strong hydrogen bonding bases

On the average in the coding sequences of 30 eucaryotic structural genes the weak hydrogen bonding, W, (A or T) or strong hydrogen bonding, S, (C or G) base in codon site 3 was chosen to be unlike its neighbors on both sides up to two sites away. This preference produced the nonrandom excess of runs W and S of length one and two and the deficit of long runs observed earlier (Blaisdell 1982). The neighbors in the different codon, 3' to codon site 3, were as important in determining the choice as were the neighbors 5' in the same codon. Every amino acid except methionine and tryptophan, of least frequent occurrence, permits choice of W or S. The persistence of this preference could explain the observation that the rate of substitution of codon site 3 in functional genes is considerably less than in synonymous pseudo genes.     

The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as measured by NMR chemical shift temperature dependencies.

In the Clostridium beijerinckii flavodoxin, the reduction of the flavin mononucleotide (FMN) cofactor is accompanied by a local conformation change in which the Gly57-Asp58 peptide bond "flips" from primarily the unusual cis O-down conformation in the oxidized state to the trans O-up conformation such that a new hydrogen bond can be formed between the carbonyl group of Gly57 and the proton on N(5) of the neutral FMN semiquinone radical [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. This interaction is thought to contribute to the relative stabilization of the flavin semiquinone and may be at least partially responsible for the substantial separation of the midpoint potentials of the two one-electron reduction steps. Through a series of amino acid substitutions, the above cited study demonstrated the critical role of the often conserved glycine residue in this process. However, it has not been directly established experimentally as to whether these substitutions brought about the changes in the midpoint potentials by altering the strength of this hydrogen-bonding interaction as proposed. In this study, the relative strengths of the FMN N(5)H.O57 hydrogen bond in wild type and the G57A, G57N, and G57T mutants were evaluated by measuring the temperature dependency of the chemical shift for the proton on N(5) of the fully reduced cofactor by 1H-15N HSQC nuclear magnetic resonance spectroscopy. Based on the established correlation between the temperature coefficient of amide protons and the strength of hydrogen bonding in small peptides, the apparent strength of the N(5)H.O57 interaction was found to depend on the properties of the side chain at position 57. The glycine residue found in the wild-type flavodoxin appears to provide the strongest interaction while the beta-branched side chain in the G57T mutant provides the weakest. A good correlation was noted between the temperature coefficients of N(5)H and the one-electron reduction potential for the ox/sq couple as well as the binding free energy of the FMN semiquinone in this group of mutants. These results provide more direct quantitative evidence that support the previous hypothesis that this conformation change and the associated formation of the hydrogen bonding interaction with N(5)H of the reduced FMN represent an important means of stabilizing the neutral semiquinone and in modulating the oxidation-reduction potentials of the flavin cofactor in this and perhaps other flavodoxins.     

Structure and energy of non-canonical basepairs: comparison of various computational chemistry methods with crystallographic ensembles

Different types of non-canonical basepairs, in addition to the Watson-Crick ones, are observed quite frequently in RNA. Their importance in the three dimensional structure is not fully understood, but their various roles have been proposed by different groups. We have analyzed the energetics and geometry of 32 most frequently observed basepairs in the functional RNA crystal structures using different popular empirical, semi-empirical and ab initio quantum chemical methods and compared their optimized geometry with the crystal data. These basepairs are classified into three categories: polar, non-polar and sugar-mediated, depending on the types of atoms involved in hydrogen bonding. In case of polar basepairs, most of the methods give rise to optimized structures close to their initial geometry. The interaction energies also follow similar trends, with the polar ones having more attractive interaction energies. Some of the C-H...O/N hydrogen bond mediated non-polar basepairs are also found to be significantly stable in terms of their interaction energy values. Few polar basepairs, having amino or carboxyl groups not hydrogen bonded to anything, such as G:G H:W C, show large flexibility. Most of the non-polar basepairs, except A:G s:s T and A:G w:s C, are found to be stable; indicating C-H...O/N interaction also plays a prominent role in stabilizing the basepairs. The sugar mediated basepairs show variability in their structures, due to the involvement of flexible ribose sugar. These presumably indicate that the most of the polar basepairs along with few non-polar ones act as seed for RNA folding while few may act as some conformational switch in the RNA.     

Contribution of hydrogen bonding to the conformational stability of ribonuclease T1.

For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins. The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1 (RNase T1). When RNase T1 folds, 86 intramolecular hydrogen bonds with an average length of 2.95 A are formed. Twelve mutants of RNase T1 [Tyr----Phe (5), Ser----Ala (3), and Asn----Ala (4)] have been prepared that remove 17 of the hydrogen bonds with an average length of 2.93 A. On the basis of urea and thermal unfolding studies of these mutants, the average decrease in conformational stability due to hydrogen bonding is 1.3 kcal/mol per hydrogen bond. This estimate is in good agreement with results from several related systems. Thus, we estimate that hydrogen bonding contributes about 110 kcal/mol to the conformational stability of RNase T1 and that this is comparable to the contribution of the hydrophobic effect. Accepting the idea that intramolecular hydrogen bonds contribute 1.3 +/- 0.6 kcal/mol to the stability of systems in an aqueous environment makes it easier to understand the stability of the "molten globule" states of proteins, and the alpha-helical conformations of small peptides.