Sequence-specific recognition of deoxyribonucleic acid. Chemical synthesis and nuclear magnetic resonance assignment of the imino protons of lambda OR3 operator deoxyribonucleic acid

Using solid-phase phosphite triester methods, we have synthesized both strands of the phage lambda OR3 DNA sequence, reannealed them, and studied the native operator duplex by high-resolution NMR at 500 MHz. At 7 degrees C the imino protons of the two terminal base pairs at each end have disappeared from the spectrum by exchange broadening. The 13 detectable imino resonances have been assigned to their respective base pairs in the duplex by using sequential nearest-neighbor NOE connectivity methods described previously. In cases where two imino protons overlap in the spectrum, spin diffusion was used to drive the cross-saturation further afield in order to produce second-order next-nearest-neighbor effects. The results show that the imino connectivity method can be used to unambiguously assign the imino proton spectrum of operator DNAs containing one to two full turns of the helix.     

Two-dimensional proton nuclear magnetic resonance investigation of the synthetic deoxyribonucleic acid decamer d(ATATCGATAT)2

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)     

Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment

The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz. The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities. Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying. Resonance from many of the base pairs suggested by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., & Matthews, B. W. (1982) Nature (London) 298, 718-723] to be involved in specific binding of the lambda phage cro repressor are well resolved.     

Proton nuclear magnetic resonance investigation of the conformation and dynamics in the synthetic deoxyribonucleic acid decamers d(ATATCGATAT) and d(ATATGCATAT)

A variety of one-dimensional proton NMR methods have been used to investigate the properties of two synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of two-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential "melting" of the A . T base pairs and by comparison of the spectra of the two decamers. Below 20 degree C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A X T and G X C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52 degrees C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8, and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5'ApT3'). The base protons (AH8, GH8, and TH6) appear to be located close (1.9-2.3 A) to sugar H2',2" protons.(ABSTRACT TRUNCATED AT 400 WORDS)     

A proton nuclear magnetic resonance study of gamma-glutamyl-amino acid cyclotransferase in human erythrocytes

Spin-echo NMR spectroscopy was shown to be a reliable technique for the monitoring of the in situ cleavage of gamma-Glu-Ala by gamma-glutamyl-amino acid cyclotransferase in whole erythrocytes and hemolysates. Of particular importance was the difference in chemical shifts between peptide resonances and those of the constituent amino acids. Using lysates of varying dilution, it was shown that the specific activity of the enzyme was not concentration-dependent, thus suggesting a lack of cytosolic low-molecular-weight-effectors or enzyme dissociation. Furthermore, the initial velocities of the reaction as a function of substrate concentration obeyed Michaelis-Menten kinetics with a Km = 2.0 +/- 0.3 mmol/l and Vmax = 137 +/- 7 mmol/h/l of cell water in 1H2O medium. Similar analysis in 2H2O medium revealed a solvent kinetic isotope effect of 1.9 +/- 0.4 at low substrate concentrations. The implications of this observation for the mechanism of the reaction are discussed. Cleavage of the peptide by a suspension of intact erythrocytes was at a rate 300 times less than the corresponding lysate flux, thus indicating the rate limitation by transport in the coupled system.     

Direct assignment of the dihydrouridine-helix imino proton resonances in transfer ribonucleic acid nuclear magnetic resonance spectra by means of the nuclear Overhauser effect

The NMR resonances from the hydrogen-bonded ring NH protons in the dihydrouridine stem of Escherichia colt tRNA1Val have been assigned by experiments involving the nuclear Overhauser effect (NOE) between adjacent base pairs. Irradiation of the 8-14 tertiary resonance produced a NOE to base pair 13. Irradiation of the CG13 ring NH produced NOEs to base pairs 12 and 14. Similarly, base pair 12 was shown to be dipolar coupled to 11 and 13, and base pair 11 was found to be coupled to 10 and 12. These sequential connectivities led to the assignment of CG13 at -13.05 ppm, UA12 at -13.84 ppm, CG11 at -12.23 ppm, and GC10 at -12.60 ppm. The results are compared with previous, less direct assignments for these four base pairs and with the expected proton positions from the crystal structure coordinates for this helix.     

Mevalonic acid is partially synthesized from amino acids in Halobacterium cutirubrum: a 13C nuclear magnetic resonance study.

13C nuclear magnetic resonance revealed an unusual pathway for the biosynthesis of lipids in Halobacterium cutirubrum and H. halobium. Mevalonic acid was not synthesized from three acetyl-coenzyme A molecules, as has been suggested previously, and the branch-methyl and methine carbons in phytanyl chains were derived from neither acetate nor glycerol. Instead, they were supplied by the degradation of amino acids, in particular of lysine. Presumably, two different types of two-carbon fragments were used simultaneously by halobacteria for the biosynthesis of mevalonate. The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.     

A proton nuclear magnetic resonance study of sulfmyoglobin cyanide

The proton nuclear magnetic resonance spectrum of sulfmyoglobin cyanide was studied at 400 MHz. The position of a methyl-group resonance at low field is consistent with a chlorin-like structure for the prosthetic group. The proton NMR spectrum of the cyanide derivative of the purified prosthetic group which decomposes upon extraction from the protein was found to be the same as that of the cyanide derivative of the prosthetic group extracted from myoglobin and a sample prepared from hemin-Cl.     

Assignment of the non-exchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods

A general method of assigning the non-exchangeable protons in the nuclear magnetic resonance spectra of small DNA molecules has been developed based upon two-dimensional autocorrelated (COSY) and nuclear Overhauser (NOESY) spectra in 2H2O solutions. Groups of protons in specific sugars or bases are identified by their scalar couplings (COSY), then connected spatially in a sequential fashion using the Overhauser effect (NOESY). The method appears to be generally applicable to moderate-sized DNA duplexes with structures close to B DNA. The self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been synthesized by the solid-phase phosphite triester technique and studied by this method. Analysis of the COSY spectrum and the NOESY spectrum leads to the unambiguous assignment of all protons in the molecule except the poorly resolved H5' and H5" resonances. The observed NOEs indicate qualitatively that, in solution, the d(C-G-C-G-A-A-T-T-C-G-C-G) helix is right-handed and close to the B DNA form with a structure similar to that determined by crystallography.     

A receptor-binding peptide from human interleukin-6: isolation and a proton nuclear magnetic resonance study.

In order to locate the receptor-binding region of human interleukin-6 (IL-6), twelve peptide fragments were prepared by digestion of IL-6 with lysylendopeptidase. A significant activity of the receptor-binding was observed only for a peptide Ile88-Lys121, although the activity was estimated at 10(4)-fold less than that of intact IL-6. Solution structure of the peptide Ile88-Lys121 was analyzed by using two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The results indicate the presence of alpha-helices in the regions Leu93-Phe106 and Glu110-Ser119. On the basis of the NMR data, we also prepared two peptides. Four-fold less binding activity than that of the peptide Ile88-Lys121 was observed for the peptide Ile88-Arg105, but no activity for the peptide Glu110-Lys121. These results suggest that the helical peptide Ile88-Arg105 composes a part of the receptor-binding region.     

Location and ion-binding of membrane-associated valinomycin, a proton nuclear magnetic resonance study

Valinomycin, incorporated in small unilamellar vesicles of perdeuterated dimyristoylphosphatidylcholine, reveals several well-resolved 1H-NMR resonances. These resonances were used to examine the location, orientation and ion-binding of membrane-bound valinomycin. The order of affinity of membrane-bound valinomycin for cations is Rb+ greater than K+ greater than Cs+ greater than Ba2+, and binding is sensitive to surface change. The exchange between bound and free forms is fast on the NMR time scale. The intrinsic binding constants, extrapolated to zero anion concentration, are similar to those determined in aqueous solution. Rb+ and K+ show 1:1 binding to valinomycin, whereas the stoichiometry of Cs+ and Ba2+ is not certain. Paramagnetic chemical shift reagents and nitroxide spin label relaxation probes were used to study the location and orientation of valinomycin in the membrane. Despite relatively fast exchange of bound cations, the time average location of the cation-free form of valinomycin is deep within the bilayer under the conditions of these experiments. Upon complexation to K+, valinomycin moves closer to the interfacial region.     

Interaction of tropomyosin and troponin T: a proton nuclear magnetic resonance study

Proton nuclear magnetic resonance (1H NMR) has been used to study the nature of the interaction between tropomyosin (TM) and troponin T (Tn-T). Resonances corresponding to the histidine residues in fragments of both TM and Tn-T can be resolved and assigned in the 1H NMR spectrum. Changes in the pH titration profiles of these resonances when the various fragments are mixed provide probes of the interaction sites between the proteins. Fragment T1 (residues 1-158) of Tn-T appears to interact weakly but specifically with fragments of TM in which the NH2-terminus (residues 1-10) is intact. While fragment CB2 (residues 71-151) of Tn-T interacts weakly (dissociation constant of 0.1-0.2 mM) with NH2-terminal fragments of TM, this appears to be nonspecific since the absence of residues 1-10 and 128-189 of TM does not affect the observed perturbations of the titration profiles of His-79 of CB2. Although a strong interaction between T1 and the COOH-terminal Cy2 fragment (residues 190-284) of TM has been previously demonstrated, no perturbation of His-276 of Cy2 or of His-7, -23, -29, or -36 of T1 was observed in a mixture of T1/Cy2. The pKa of His-276 was also not affected in a mixture of Cy1/Cy2 (where Cy1 is residues 1-189 of TM) but was significantly decreased in the ternary complex T1/Cy1/Cy2. The importance of residues 1-70 of Tn-T in its binding to TM is illustrated by the specificity it confers on the T1/Cy1 interaction and by the absence of His-276 perturbation in the mixture CB2/Cy1/Cy2.     

Conformational variations in d(TGACGTCA) and its reverse sequence d(ACTGCAGT): a joint circular dichroism and nuclear magnetic resonance study

Circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques have been used to characterize the structural properties of the two self-complementary DNA octamers d(TGACGTCA) (I) and d(ACTGCAGT) (II). These display as distinctive features reverse sequences and central steps CpG and GpC, respectively. CD experiments lead to B-form DNA spectra characterized by larger magnitude signals in the case of octamer (I). NMR COSY spectra indicate that in the two octamers all the residues are predominantly south, S, (2'-endo) sugar conformation. NMR NOESY spectra show most of the glycosidic angles confined in the range predicted for B-form DNA although important heterogeneity is noticed along the chains, more pronounced in the case of octamer (I). Both the increase of north, N, (3'-endo) sugar conformation and P (pseudorotation phase angle) deviation from its standard B-form DNA value (162 degrees) express local sequence dependent structure distortions, remarkably visible in CpG step of octamer (I) and agreeing with NOESY cross-peaks intensities. Results interpreted according to Calladine's rules indicate higher cross-chain strains in octamer (I) than in octamer (II). All together, we find evidence to support for octamer (I) in solution local structures with A-DNA properties likely dictated by the central CpG step. Such structures could be involved in the DNA recognition by proteins and anticancerous drugs.     

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipid vesicles: A proton nuclear magnetic resonance study

The kinetics of Ca²⁺-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125–300 Å. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates on Ca²⁺ as well as an vesicle concentration. For vesicle concentrations in the range of 3 · 10^?6–10^?5 M and Ca²⁺ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1–10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.     

Amino acid analysis of angiotensin I by proton nuclear magnetic resonance spectroscopy

The chemical shifts of the isoleucine and histidine protons of angiotensin I were assigned and the chemical shifts of the protons of the other amino acids in the peptide were confirmed at a field strength of 400 MHz. These chemical shift assignments were used to determine the amino acid composition of angiotensin I. These data were then compared to the amino acid composition which was determined by chromatographic analysis of the peptide hydrolysate. The results obtained by the chromatographic method were similar to those obtained by the NMR method. The standard deviations of the results were similar, indicating that these methods are equally precise. The major advantages of the NMR method are that it permits the recovery of the peptide after completion of the analysis and improves the quantitation of amino acids which are either partially destroyed by the hydrolysis procedure or require special derivatization methods for detection and quantitation.     

Bleomycin-A2 complexes with poly(dA--dT): a proton nuclear magnetic resonance study of the nonexchangeable hydrogens

The mRNA coding for uteroglobin, a progesterone-induced uterine protein, has been partially purified from 4-day pregnant rabbit uterus. Double-stranded DNA synthesized from the partially purified mRNA preparation was inserted into the Pst I site of pBR 322. Bacterial transformants containing uteroglobin DNA sequences were identified by their ability to enrich for uteroglobin mRNA on hybridization with total uterine poly A-RNA. The identity of one recombinant was confirmed unambiguously by matching its nucleotide sequence with the amino acid sequence of the uteroglobin polypeptide.