'Interactive' recognition in EcoRI restriction enzyme-DNA complex.

A solution study of interaction between DNA and EcoRI restriction enzyme shows that there is a definite distortion of DNA in the specific recognition complexes but no measurable DNA distortion in the non-specific interaction.     

Drug-DNA interactions: spectroscopic and footprinting studies of site and sequence specificity of elliptinium.

The binding of the antitumoral ellipticine derivative 2-methyl-9-hydroxyellipticinium acetate (elliptinium; NMHE) to DNA was analyzed by the combined use of DNase I footprinting and spectroscopic methods. Using two fragments of pBR322 DNA, five discrete NMHE binding sites of 5-7 protected base pairs (bp) were detected by footprinting at 4 degrees C on the analyzed regions. These corresponded to alternating pyrimidines and purines. The inactive derivative 2-methyl ellipticinium acetate L(NME) lacking a hydroxy group failed to demonstrate DNA protection even at low temperature. Ultraviolet-absorption and 1H-nmr analysis was performed using two autocomplementary octanucleotides d(TGACGTCA) (I) and d(ACTGCAGT) (II). The uv-absorption titrations resulted in an intercalative binding mode for NMHE in the oligomers. Analysis of the derived biphasic Scatchard plots yielded two binding sites corresponding to approximately 6-bp and 2-bp sizes and characterized by apparent association constants K1 approximately 10(8) M-1 and K2 approximately 10(6) M-1, respectively. The 1H-nmr analysis of exchangeable (imino) protons and nonexchangeable protons performed in the one- and two-dimensional modes confirmed the intercalation of NMHE, and further revealed the existence of multiple sites on DNA. Assuming that imino resonance line width concerned the sole kinetic effects, 10-ms order lifetimes were estimated for the drug-oligonucleotide complexes at 7 degrees C, pH 7, and 0.1 ionic strength. Finally, examination of every drug-DNA spectra in the light of the footprinting results indicated that there was a preference for binding of NMHE to the CpG (octamer I) and TpG (octamers I and II) steps.     

NMR studies of water-gas interactions

Analysis of published data concerning the solubility of different gases in water as dependent on temperature was carried out. These dependences could be described by mono-or bi-exponential functions. Solubilities of nitrogen and oxygen are additive and depend on their percentage in the atmosphere over the liquid. The temperature dependence of oxygen and nitrogen dissolved in water corresponds to that in the atmospheric air. Measurements of water spin-lattice relaxation times, changing with the concentration of dissolved paramagnetic oxygen, showed that oxygen could be substantially but not completely eliminated by saturation with any gas. The best method is the contact with a water-immiscible liquid of gas capacity higher than water. However, all this leads to an unstable state of a gas-water system, converging to equilibrium.     

An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity

To test their structural and functional similarity, hybrids were constructed between EcoRI and RsrI, two restriction endonucleases recognizing the same DNA sequence and sharing 50% amino acid sequence identity. One of the chimeric proteins (EERE), in which the EcoRI segment His147-Ala206 was replaced with the corresponding RsrI segment, showed EcoRI/RsrI-specific endonuclease activity. EERE purified from inclusion bodies was found to have approximately 100-fold weaker activity but higher specific DNA binding affinity, than EcoRI. Increased binding is consistent with results of molecular dynamics simulations, which indicate that the number of hydrogen bonds formed with the recognition sequence increased in the chimera as compared to EcoRI. The success of obtaining an EcoRI-RsrI hybrid endonuclease, which differs from EcoRI by 22 RsrI-specific amino acid substitutions and still preserves canonical cleavage specificity, is a sign of structural and functional similarity shared by the parental enzymes. This conclusion is also supported by computational studies, which indicate that construction of the EERE chimera did not induce substantial changes in the structure of EcoRI. Surprisingly, the chimeric endonuclease was more toxic to cells not protected by EcoRI methyltransferase, than the parental EcoRI mutant. Molecular modelling revealed structural alterations, which are likely to impede coupling between substrate recognition and cleavage and suggest a possible explanation for the toxic phenotype.     

SOS induction as an in vivo assay of enzyme-DNA interactions

We have constructed strains which are convenient and sensitive indicators of DNA damage and describe their use. These strains utilize an SOS::lac Z fusion constructed by Kenyon and Walker [Proc. Natl. Acad. Sci. USA 77 (1980) 2819-2823] and respond to DNA damage by producing beta-galactosidase. They can be used to characterize restriction systems and screen for restriction endonuclease mutants. Applications include the study of other enzymes involved in DNA metabolism, such as DNA methyltransferases, topoisomerases, recombinases, and DNA replication and repair enzymes.     

Rational engineering of sequence specificity in R.MwoI restriction endonuclease

R.MwoI is a Type II restriction endonucleases enzyme (REase), which specifically recognizes a palindromic interrupted DNA sequence 5'-GCNNNNNNNGC-3' (where N indicates any nucleotide), and hydrolyzes the phosphodiester bond in the DNA between the 7th and 8th base in both strands. R.MwoI exhibits remote sequence similarity to R.BglI, a REase with known structure, which recognizes an interrupted palindromic target 5'-GCCNNNNNGGC-3'. A homology model of R.MwoI in complex with DNA was constructed and used to predict functionally important amino acid residues that were subsequently targeted by mutagenesis. The model, together with the supporting experimental data, revealed regions important for recognition of the common bases in DNA sequences recognized by R.BglI and R.MwoI. Based on the bioinformatics analysis, we designed substitutions of the S310 residue in R.MwoI to arginine or glutamic acid, which led to enzyme variants with altered sequence selectivity compared with the wild-type enzyme. The S310R variant of R.MwoI preferred the 5'-GCCNNNNNGGC-3' sequence as a target, similarly to R.BglI, whereas the S310E variant preferentially cleaved a subset of the MwoI sites, depending on the identity of the 3rd and 9th nucleotide residues. Our results represent a case study of a REase sequence specificity alteration by a single amino acid substitution, based on a theoretical model in the absence of a crystal structure.     

The role of peripheral interactions in chymosin specificity

Kinetic parameters for the splitting of model peptide substrates and chi-casein with chymosin have been interpreted on the basis of the three-dimensional structure of chymosin. Model peptide substrates contain a fragment of the chi-casein sequence in the region of the bond Phe-105--Met-106 splitted with the enzyme. It was shown that the possible reason of the enormous milk-clotting efficiency of chymosin may be partly associated with the electrostatic interaction of the positive charged segment 98-102 (His-Pro-His-Pro-His) of the substrate and outer loop of the enzyme which contains Glu-245, Asp-247, Asp-249, Asp-251.     

Non-additivity of sequence-specific enzyme-DNA interactions in the EcoRI DNA methyltransferase.

We describe a novel strategy to characterize protein-DNA interactions involving monomeric enzymes such as DNA methyltransferases (Mtases). This strategy is applied to our investigation of the EcoRI DNA Mtase, which binds its double stranded recognition site 5'-G-AATTC-3' and methylates the central adenosine of each strand using S-adenosyl-L-methionine as the methyl donor. We show that prior methylation of adenosine in either strand does not perturb catalysis. In contrast, substrates substituted with deoxyinosine at either guanosine position (T-BMI5 and TI5-BM) show the minor groove residing N2 amino group of both guanosines contribute to DNA recognition since specificity constants for the modified substrates are reduced 13 and 39 fold. Similar analysis of a substrate containing deoxyinosine at both positions (TI5-BMI5) clearly shows that some communication occurs between the sites. To determine the extent to which structural changes in the DNA alone contribute to this lack of additivity, we performed DNA melting analysis of the singly and doubly substituted substrates, and also found non-additivity. Although our functional and structural analyses suggest that deoxyinosine incorporation causes long range conformational effects, the similarity of KmAdoMet for all substrates suggests that no large-scale structural changes occur in the Mtase-DNA-AdoMet complex. Our results support the following conclusions: 1) The non-additivity shown in this system contrasts with the widespread demonstration of additivity involving repressors [Lehming et al., 1990; Takeda et al., 1989; Ebright et al., 1987], suggesting that sequence discrimination by enzymes may involve more complex mechanisms. Further, this non-additivity precludes quantitative assignment of individual interactions and we suggest that future analyses of this and related enzyme systems with base analogs include detailed information about the long range structural consequences of individual substitutions. 2) Although TI5-BM and T-BMI5 are shown to be radically different by thermodynamic analysis, the similar specificity constants with the Mtase suggest that the underlying structural differences (e.g., altered helical parameters of the DNA) are not critical for sequence-recognition. 3) The significance of minor groove Mtase-DNA interactions to specificity is confirmed.     

The conformation of tetrafluorinated methyl galactoside anomers: crystallographic and NMR studies

The first single-crystal X-ray diffraction study of tetrafluorinated monosaccharide derivatives is presented. Both α- and β-methyl 2,3-dideoxy-2,2,3,3-tetrafluoro-d-galactopyranoside anomers adopt the ⁴C₁ conformation. The values for the C1–O1 and C1–O5 bond lengths and the O5–C1–O1–CH₃ dihedral angles are in line with what can be expected from the anomeric and exo-anomeric effects. The chair conformations are slightly distorted, presumably due to repulsion between 1,3-diaxial C–O and C–F bonds. The asymmetric unit of both compounds contains up to three independent molecules, which differ in the conformation of the hydroxymethyl group (including in one case a ‘forbidden’ gg rotamer). The molecular packing of the β-anomer shows a clear segregation between fluorinated and hydrophilic domains, while for the α-anomer the regions of fluorine segregation are broken by interleafing of OMe groups. There is one close OH?F contact, which is likely to arise from the crystal packing. NMR studies show that the two anomers also adopt a ⁴C₁ conformation in solution (D₂O, CDCl₃).     

Sodium-23 NMR studies of cation-DNA interactions

Sodium-23 NMR has been used to study the extent to which monovalent cations associate with double stranded DNA in aqueous solution (28°C, pH = 7.5). On the basis of the two site model for rapid exchange the ²³Na linewidth can be related to the fraction of sodium ions associated with DNA. To test the applicability to this system of the condensation model for the association of small counterions with polyelectrolytes, the concentration dependence of the sodium linewidth has been determined by making additions of NaCl to solutions of tetraethyl or tetrabutylammonium DNA. ([P], the DNA phosphate concentration was about 0.02M). The resulting titration curves extend over a wide range of the ratio [Na]/[P] (0.3–30). When [Na]/[P] ? 3 only sodium is associated, and the extent to which it compensates the charges on DNA does not vary with the addition of salt, at least until [Na]/[P] ≈ 30, the highest concentration examined. When [Na]/[P] ? 3 the tetraalkylammonium species is also associated with DNA; an equation has been derived to account for the effect on the ²³Na linewidth of the competition between sodium and another monovalent cation. Based on the assumption that the fraction of uncompensated charge remaining on DNA after the condensation of both species is constant, this equation fits all the linewidth data if the charge fraction is in the range 0.25 ± 0.10. The value required by the condensation model for DNA in the presence of monovalent counterions is ξ^?1 = 0.24. The reasonable agreement between experimental and theoretical values of the charge fraction and its invariance with respect to large variations in the concentration of added salt indicate that even in moderately concentrated solutions of DNA, the association of sodium can usefully be described in terms of the condensation model. If the theoretical value of the charge fraction is assumed, it follows from fitting the titration curves that the approximate relative affinities for DNA of Na⁺, Et₄N⁺, and Bu₄N⁺ are in the ratio 20:5:1, and the transverse relaxation rate of condensed sodium is 180 ± 10 s^?1.     

NMR studies of the antibody-bound conformation of a carbohydrate-mimetic peptide

Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed at 800 MHz to investigate the bound conformation of the hexapeptide DRPVPY, a functional molecular mimic of the group A Streptococcus cell-wall polysaccharide. The hexapeptide binds to the monoclonal antibody SA-3, mimicking the branched trisaccharide repeating unit, L-Rha-alpha-(1 --> 2)-(D-GlcNAc-beta-(1 --> 3))-alpha-L-Rha (Rha, rhamnose; GlcNAc, N-acetylglucosamine). The peptide adopts a tight turn conformation with close contacts between the side chains of valine and tyrosine. Relaxation network editing experiments (QUIET-NOESY) were used to confirm the validity of the observed contacts and to evaluate the presence of spin diffusion pathways. Saturation transfer difference (STD-NMR) experiments with selective saturation of protein resonances revealed enhancements of many of the peptide resonances due to close contacts between the peptide and the protein within the antibody combining site.     

Solution NMR studies of peptide-lipid interactions in model membranes

Abstract

Many important processes in life take place in or around the cell membranes. Lipids have different properties regarding their membrane-forming capacities, their mobility, shape, size and surface charge, and all of these factors influence the way that proteins and peptides interact with the membrane. In order for us to correctly understand these interactions, we need to be able to study all aspects of the interplay between lipids and peptides and proteins. Solution-state NMR offers a somewhat unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating peptide-lipid interaction, and special attention is given to the various membrane mimetics that are used to model the membrane. Examples from the field of cell-penetrating peptides and their lipid interactions will be given. The importance of studying lipid and peptide dynamics, which reflect on the effect that peptides have on bilayers, is highlighted, and in this respect, also the need for realistic membrane models.     

Solution NMR studies of peptide-lipid interactions in model membranes

Many important processes in life take place in or around the cell membranes. Lipids have different properties regarding their membrane-forming capacities, their mobility, shape, size and surface charge, and all of these factors influence the way that proteins and peptides interact with the membrane. In order for us to correctly understand these interactions, we need to be able to study all aspects of the interplay between lipids and peptides and proteins. Solution-state NMR offers a somewhat unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating peptide-lipid interaction, and special attention is given to the various membrane mimetics that are used to model the membrane. Examples from the field of cell-penetrating peptides and their lipid interactions will be given. The importance of studying lipid and peptide dynamics, which reflect on the effect that peptides have on bilayers, is highlighted, and in this respect, also the need for realistic membrane models.     

On the role of peripheral interactions in specificity of chymosin

Chymosin is distinguished by a high level of milk-clotting activity which is the consequence of the specific cleavage of the Phe(105)-Met(106) bond of kappa-casein. Based on modelling considerations it was proposed that milk-clotting activity of chymosin is associated with electrostatic interactions of a charged segment His-Pro-His-Pro-His (98-102) of casein and the outer loop of the enzyme containing Glu-244,Asp-246 and Asp-248.     

Deuterium NMR relaxation studies of peptide-lipid interactions.

A unique model membrane system composed of a synthetic amphiphilic peptide (Lys2-Gly-Leu16-Lys2-Ala-amide) and a specifically labeled phospholipid (1,2-[7,7-2H2]dipalmitoyl-sn-glycero-3-phosphocholine) has been studied by 2H NMR, using inversion recovery, quadrupolar echo, and modified Jeener-Broekaert sequences, from 213 to 333 K, at molar peptide concentrations of 0, 2, 4, and 6%. Analysis of the experiments, employing a density matrix treatment based on the stochastic Liouville equation, revealed information about the dynamic organization of the lipid in the model membrane system, whose phase behavior has been determined previously [Huschilt et al. (1985) Biochemistry 24, 1377-1386]. The dynamic organization is described in terms of segmental and molecular order parameters and in terms of correlation times corresponding to both internal and overall lipid motions. In the liquid crystalline phase, the molecular order parameter, SZZ, was observed to decrease slightly upon addition of peptide while the conformational order parameter corresponding to the seventh segment, SZ'Z', did not change for any concentration of peptide. In general, the gauche-trans isomerization rate in the middle of the chain was not observed to change upon peptide addition, whereas the whole body reorientational correlation times (tau R parallel and tau R perpendicular) increased by nearly an order of magnitude. The anisotropy ratio (tau R perpendicular/tau R parallel) decreased with peptide added. An additional motion which involves a jump about the axis of the sn-2 chain is also observed to be slowed down significantly in the presence of peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of the conformation of the natural sweetener rebaudioside A

Rebaudioside A is a natural sweetener from Stevia rebaudiana in which four β-d-glucopyranose units are attached to the aglycone steviol. Its ¹H and ¹³C NMR spectra in pyridine-d₅ were assigned using 1D and 2D methods. Constrained molecular dynamics of solvated rebaudioside using NMR constraints derived from ROESY cross peaks yielded the orientation of the β-d-glucopyranose units. Hydrogen bonding was examined using the temperature coefficients of the hydroxyl chemical shifts, ROESY and long-range COSY spectra, and proton-proton coupling constants.     

A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity

We have mutated the monomer-monomer interface of the restriction endonuclease EcoRI in order to destabilize the homodimer and to stabilize heterodimers. Mutations of Leu158 to charged amino acid residues result in strong destabilization of the dimer. The largest effect was detected for the L158D mutant which is monomeric even at higher concentrations. It unspecifically degrades DNA by cleaving both single strands independently every 15 nucleotides on the average. Although cleavage is reproducible, it is not determined by nucleotide sequence but by general properties like conformation or deformability as has been found for other unspecific nucleases. Mutations of Ile230, which is in direct contact with Leu158 of the other subunit, cause structural changes with the loss of about ten percent alpha-helix content, but interfere only marginally with homodimerization and double strand cleavage. Again the mutation to aspartate shows the strongest effects. Mixtures of single mutants, one containing aspartate at one of the two positions and the other lysine at the corresponding position, form heterodimers. These are mainly stabilized compared to the homodimers by re-establishment of the wild-type hydrophobic interaction at the not mutated residues while an interaction of aspartate and lysine seems energetically unfavorable in this structural context.