"Asymmetric" opening reaction mechanism of Z-DNA base pairs: a hydrogen exchange study

With the tritium-Sephadex method, the hydrogen-exchange kinetics of the five NH protons of guanine and cytosine residues in Z-form poly(dG-dC) X poly (dG-dC) were measured as a function of temperature and catalyst concentration. Over the measured temperature range from 0 to 34 degrees C, two classes of protons with constant amplitudes are found. The three protons of the fast class, which were assigned to the guanine amino and imino protons, have an exchange half-time in the minute time range (at 20 degrees C the half-time is 2.5 min) and an activation energy of 18 kcal mol-1. Since these two types of protons exchange at the same rate in spite of their grossly different pK values, the exchange of these protons must be limited by the same nucleic acid conformational change. The two cytosine amino protons of the slow class are especially slow with exchange half-times in the hour time range (at 20 degrees C the exchange half-time is 1 h) and the activation energy is 20 kcal mol-1. The exchange of these two protons is not limited by some nucleic acid conformational change as shown by the marked exchange acceleration of these protons upon addition of 0.2 M imidazole. In addition, we have also reexamined the hydrogen-deuterium exchange kinetics of the amino protons of guanosine cyclic 2',3'-monophosphate by a spectral difference method using a stopped-flow spectrophotometer. The measured kinetic process is monophasic with a rate constant of 3 s-1 at 20 degrees C, which is in the same range as the predicted rate constant of the guanine amino protons.(ABSTRACT TRUNCATED AT 250 WORDS)     

The conformation of the d(ACCCGGGT) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)2 have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D2O at 12 degrees C. The observed NOE's between the base protons and their own H2' protons and between the base protons and the H2' protons of the 5' adjacent nucleotide and the observed coupling constants between the deoxyribose 1' and 2',2' protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.     

A hydrogen-deuterium exchange study of the amide protons of polymyxin B by nuclear-magnetic-resonance spectroscopy.

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and gamma-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two beta turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.     

Identification of spin diffusion pathways in proteins by isotope-assisted NMR cross- relaxation network editing

A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE = Double INEPT Edited), is proposed. It is based on the selectiveinversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sortsout the spin diffusion paths according to the principal mediators, either the CH/CH3 or theCH2 protons. This is useful in the structure refinement process, as it enables proper alignmentof the aliphatic protons in the vicinity of NH protons.     

Properties of pseudouridine N1 imino protons located in the major groove of an A-form RNA duplex.

The exchangeable N1 imino protons of two pseudouridine (psi) bases located at adjacent internal positions within an undecamer RNA duplex (5'AUAC psi psi ACCUG/3'UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The psi N1 imino protons of these residues (which are not involved in interstrand Watson-Crick hydrogen bonding) are protected from chemical exchange with the solvent water and thus are observable in the proton NMR spectrum in H2O (1). These protons will exchange readily at increased pH values or upon thermal denaturation of the duplex. The longitudinal (T1) relaxation times of the psi N1 imino protons in 100 mM NaCl or in 10 mM MgCl2 and 100 mM NaCl are approximately two-fold faster than those of the psi N3 imino protons which are involved in Watson-Crick base pairing. With the addition of spermidine, the psi N1 imino protons become readily exchangeable at a temperature some 20 degrees C below the melting temperature of the duplex.     

A stopped-flow H-D exchange kinetic study of spermine-polynucleotide interactions.

The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies.     

Two-dimensional 1H-NMR investigation of ribonuclease T1. Resonance assignments, secondary and low-resolution tertiary structures of ribonuclease T1

Ribonuclease T1 was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequence-specific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and C beta protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects.     

Dynamics and binding mode of Hoechst 33258 to d(GTGGAATTCCAC)2 in the 1:1 solution complex as determined by two-dimensional 1H NMR.

We have investigated the interaction of the bisbenzimidazole derivative Hoechst 33258 with the self-complementary dodecadeoxynucleotide duplex d(GTGGAATTCCAC)2 using one-dimensional (1D) and two-dimensional (2D) proton nuclear magnetic resonance (1H NMR) spectroscopy. To monitor the extent of complex formation, we used the imino proton region of the 1D 1H NMR spectra acquired in H2O solution. These spectra show that the DNA duplex loses its inherent C2v symmetry upon addition of the drug, indicating that the two molecules form a kinetically stable complex on the NMR time scale (the lifetime of the complex has been measured to be around 450 ms). We obtained sequence-specific assignments for all protons of the ligand and most protons of each separate strand of the oligonucleotide duplex using a variety of homonuclear 2D 1H NMR experiments. The aromatic protons of the DNA strands, which are symmetrically related in the free duplex, exhibit exchange cross peaks in the complex. This indicates that the drug binds in two equivalent sites on the 12-mer, with an exchange rate constant of 2.2 +/- 0.2 s-1. Twenty-five intermolecular NOEs were identified, all involving adenine 2 and sugar 1' protons of the DNA and protons in all four residues of the ligand, indicating that Hoechst 33258 is located in the minor groove at the AATT site. Only protons along the same edge of the two benzimidazole moieties of the drug show NOEs to DNA protons at the bottom of the minor groove. Using molecular mechanics, we have generated a unique model of the complex using distance constraints derived from the intermolecular NOEs. We present, however, evidence that the piperazine group may adopt at least two locally different conformations when the drug is bound to this dodecanucleotide.     

Study of the structure of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA) .pd(TGTTTGGC) with covalently bound 10-(2-hydroxyethyl)phenazine in an aqueous solution by 2D-1H-NMR spectroscopy]

The spatial structure of duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) having a N-(2-oxyethyl)-phenazinium residue covalently linked with the 5'-terminal phosphate of the heptanucleotide was studied by means of one- and two-dimensional 1H-NMR spectroscopy. The resonances of phenazinium protons, ethylenediamine linker protons, as well as, oligonucleotide H5/H6/H8/CH3 base protons and H1',H2'a, H2'b, H3', H4' deoxyribose protons have been assigned by means of 1H-COSY, 1H-NOESY and 1H-13C-COSY. The presence of the phenazine residue in duplex causes an additional imino proton signal of the terminal (G-7).(C-1) base pair, suggesting a higher stability of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) as compared to the unmodified duplex pd(CCAAACA).pd(TGTTTGGC). Analysis of NOE interactions between protons of the dye and the oligonucleotides show the phenazinium polycyclic system to intercalate between G-7 and C-8 residues of the octanucleotide.     

Strategy for trapping intermediates in the folding of ribonuclease and for using 1H-nmr to determine their structures

The major unfolded form of ribonuclease A is known to show well-populated structural intermediates transiently during folding at 0°–10°C. We describe here how the exchange reaction between D₂O and peptide NH protons can be used to trap folding intermediates. The protons protected from exchange during folding can be characterized by ¹H-nmr after folding is complete. The feasibility of using ¹H-nmr to resolve a set of protected peptide protons is demonstrated by using a specially prepared sample of ribonuclease S in D₂O in which only the peptide protons of residues 7–14 are in the ¹H-form. All eight of these protected peptide protons are H-bonded. Resonance assignments made on isolated peptides containing these residues have been used to identify the protected protons. Other sets of protected protons trapped in the ¹H-form can also be isolated by differential exchange, using either ribonuclease A or S. Earlier model compound studies have indicated that H-bonded folding intermediates should be unstable in water unless stabilized by additional interactions. Nevertheless, peptides derived from ribonuclease A that contain residues 3–13 do show partial helix formation in water at low temperatures. We discuss the possibility that specific interactions between side chains can stabilize short α-helixes by nucleating the helix, and that specific interactions may also define the helix boundaries at early stages in folding.     

The Conformation of the d(ACCCGGGT) Duplex in Aqueous Solution

Abstract

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)₂ have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D₂O at 12°C. The observed NOE's between the base protons and their own H2′ protons and between the base protons and the H2′ protons of the 5′adjacent nucleotide and the observed coupling constants between the deoxyribose 1′ and 2′,2″ protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.     

1H-NMR studies of [Sar1]angiotensin II conformation by nuclear Overhauser effect spectroscopy in the rotating frame (ROESY): clustering of the aromatic rings in dimethylsulfoxide

The conformational properties of the octapeptide [Sar1]ANG II in dimethylsulfoxide-d6 were investigated by rotating frame nuclear Overhauser effect spectroscopy (ROESY). Interresidue ROESY interactions were observed between Tyr ortho and Phe ring protons, between Phe ring and Pro C gamma protons, and also between His C alpha and Pro C delta protons. A weak connectivity was also observed between the Sar N-CH3 protons and a Tyr ortho proton. Intraresidue interactions between alpha and beta protons in Tyr, His and Phe indicated restricted rotation for the side-chains of the three aromatic residues. These findings suggest that [Sar1]ANG II takes up a folded conformation in DMSO in which the three aromatic rings form a cluster. Connectivities between the His C alpha proton and the two Pro C delta protons illustrated a preferred conformation for angiotensin II in DMSO in which the His-Pro bond exists as the trans isomer. The NMR spectroscopic evidence is consistent with the presence of a Tyr charge relay system in the biologically active conformation of angiotensin II and with the postulated role of the Tyr hydroxyl group in angiotensin II for receptor activation.     

Biosynthèse de cellulose bactérienne à pertir de glycérol sélectivement deutérié en position 1, 2, ou 3: Etude par résonance magnétique nucléaire

Glycerol specifically deuterated at C-1, C-2, or C-3 was prepared and used for the biosynthesis of bacterial cellulose with Acetobacter xylinum.The material obtained were converted into glucitol hexaacetate and analyzed by 250-MHz nuclear magnetic resonance and mass spectrometry. The spectra indicated that the protons of the C-3 position of the starting glycerol were incorporated as substituents of the C-6 and C-1 positions of the cellulose. Similarly, protons of the C-2 and C-5 positions of the cellulose came essentially from water and the protons bonded at the C-3 and C-4 positions of the cellulose from protons bonded to C-1 of the starting glycerol.     

Sequence-dependent conformations of DNA duplexes: the TATA segment of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

The base and sugar protons of the d(G-G-T-A-T-A-C-C) duplex have been assigned from two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements in D₂O solution at 25°C. The nucleic acid protons have been assigned from NOEs between protons on adjacent bases on the same and partner strands, as well as from NOEs between the base protons and their own and 5′-flanking H1′, H2′, H2″, H3′, and H4′ sugar protons. These assignments are confirmed from coupling constant and NOE connectivities within the sugar protons of a given residue. Several of these NOEs exhibit directionality and demonstrate that the d(G-G-T-A-T-A-C-C) duplex is a right-handed helix. The relative magnitude of the NOEs between the base protons and the sugar H2′ protons of its own and 5′-flanking sugar demonstrate that the TATA segment of the d(G-G-T-A-T-A-C-C) duplex adopts a B-DNA type helix geometry in solution, in contrast to the previous observation of a A-type helix for the same octanucleotide duplex in the crystalline state.     

Two-dimensional NMR studies on the anthramycin-d(ATGCAT)2 adduct

Two-dimensional NMR experiments were performed on the adduct of anthramycin with d(ATGCAT)2 to obtain the assignments of the nucleotide base and sugar protons as well as the anthramycin protons. Anthramycin is covalently attached to a guanine 2-amino group, forming the d(ATamGCAT).d(ATGCAT) modified duplex. The anthramycin protons in the minor groove exhibit NOEs to several nucleotide protons. The network of anthramycin-nucleotide NOEs and the measurement of the 10-Hz coupling constant between the anthramycin H11 and H11a protons shows that anthramycin is covalently attached as the S stereoisomer at the anthramycin C11 position with the side chain of anthramycin oriented toward the 5' end of the modified strand. The NOE data show that the anthramycin-modified duplex is in a right-handed conformation with all bases in an anti conformation. Analysis of the J1'-2' coupling constants for the resolved H1' resonances shows that the S-type conformation of the sugars is highly preferred.     

Selective 31P(1H) nuclear Overhauser effect study on the polar headgroup conformation of phospholipids in micelles in organic solvents

The polar headgroup structure of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in inverted micelles in chloroform or benzene was investigated by the selective 31P(H) nuclear Overhauser effect (NOE). In the frequency dependence of the 31P(1H) NOE, PC micelles in CDCl3 showed two maxima. The larger maximum was located at the resonance of the glycerol-CH2OP protons and the smaller at the resonance of the N-methyl protons. In PC/PE mixed micelles in C6D6, both PC and PE showed three maxima which were located at the resonance of the CH2OP protons, the N-methyl protons and the amino protons in the frequency dependence of the 31P-NOE. The N-methyl protons of PC and the amino protons of PE were closely spaced to the phosphate groups of neighboring lipid molecules. The polar headgroups of PC and PE in the mixed micelles were concluded to lie in the plane perpendicular to the molecular axes. The frequency dependence of the 31P(H) NOE for PS micelles in C6D6 showed the maxima at the resonances of the amino protons and the CH2OP protons. The polar headgroups of PS molecules were not extended parallel to the molecular axes in the inverted micelles.     

Proton and deuteron relaxation of muscle water over wide ranges of resonance frequencies.

The spin-lattice relaxation time (T1) of water protons in mouse muscle was studied from 10(4) to 10(8) Hz at several temperatures, and the deuteron T1 of muscle water was studied from 2.0 X 10(3) to 1.54 X 10(7) Hz at several temperatures. Proton T1's of muscle and brain water with different D2O contents were measured at 25 degrees C and 35 MHz. From the results of variable frequency and temperature measurements and the data of isotope substitution, it is concluded that the major relaxation mechanism for the protons in muscle water is the intermolecular dipolar interaction between the protons of the macromolecules and the protons of the water molecules in the hydration layer. It is also suggested that the relaxation of deuterons can be accounted for a very small fraction of water molecules directly hydrogen-bonded to the macromolecules.     

Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring 1H nuclear magnetic resonances in clostridial-type ferredoxins.

We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin.     

The absorption of protons with specific amino acids and carbohydrates by yeast

1. Proton uptake in the presence of various amino acids was studied in washed yeast suspensions containing deoxyglucose and antimycin to inhibit energy metabolism. A series of mutant strains of Saccharomyces cerevisiae with defective amino acid permeases was used. The fast absorption of glycine, l-citrulline and l-methionine through the general amino acid permease was associated with the uptake of about 2 extra equivalents of protons per mol of amino acid absorbed, whereas the slower absorption of l-methionine, l-proline and, possibly, l-arginine through their specific permeases was associated with about 1 proton equivalent. l-Canavanine and l-lysine were also absorbed with 1-2 equivalents of protons. 2. A strain of Saccharomyces carlsbergensis behaved similarly with these amino acids. 3. Preparations of the latter yeast grown with maltose subsequently absorbed it with 2-3 equivalents of protons. The accelerated rate of proton uptake increased up to a maximum value with the maltose concentration (K(m)=1.6mm). The uptake of protons was also faster in the presence of alpha-methylglucoside and sucrose, but not in the presence of glucose, galactose or 2-deoxyglucose. All of these compounds except the last could cause acid formation. The uptake of protons induced by maltose, alpha-methylglucoside and sucrose was not observed when the yeast was grown with glucose, although acid was then formed both from sucrose and glucose. 4. A strain of Saccharomyces fragilis that both fermented and formed acid from lactose absorbed extra protons in the presence of lactose. 5. The observations show that protons were co-substrates in the systems transporting the amino acids and certain of the carbohydrates.     

ATP-synthase of Rhodobacter capsulatus: coupling of proton flow through F0 to reactions in F1 under the ATP synthesis and slip conditions

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F0F1 and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F0F1. The proton transfer through F0F1 after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATP-synthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.