High field 1H-NMR analysis of the 1:1 intercalation complex of the antitumor agent mitoxantrone and the DNA duplex [d(CpGpCpG)

Complete 1H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]2. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of 1H and 13C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and 31P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

Crystallographic structure of the K intermediate of bacteriorhodopsin: conservation of free energy after photoisomerization of the retinal

The K state, an early intermediate of the bacteriorhodopsin photocycle, contains the excess free energy used for light-driven proton transport. The energy gain must reside in or near the photoisomerized retinal, but in what form has long been an open question. We produced the K intermediate in bacteriorhodopsin crystals in a photostationary state at 100K, with 40% yield, and determined its X-ray diffraction structure to 1.43 A resolution. In independent refinements of data from four crystals, the changes are confined mainly to the photoisomerized retinal. The retinal is 13-cis,15-anti, as known from vibrational spectroscopy. The C13=C14 bond is rotated nearly fully to cis from the initial trans configuration, but the C14-C15 and C15=NZ bonds are partially counter-rotated. This strained geometry keeps the direction of the Schiff base N-H bond vector roughly in the extracellular direction, but the angle of its hydrogen bond with water 402, that connects it to the anionic Asp85 and Asp212, is not optimal. Weakening of this hydrogen bond may account for many of the reported features of the infrared spectrum of K, and for its photoelectric signal, as well as the deprotonation of the Schiff base later in the cycle. Importantly, although 13-cis, the retinal does not assume the expected bent shape of this configuration. Comparison of the calculated energy of the increased angle of C12-C13=C14, that allows this distortion, with the earlier reported calorimetric measurement of the enthalpy gain of the K state indicates that a significant part of the excess energy is conserved in the bond strain at C13.     

High Field 1H-NMR Analysis of the 1:1 Intercalation Complex of the Antitumor Agent Mitoxantrone and the DNA Duplex [d(CpGpCpG)]2

Abstract

Complete ¹H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]₂. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of ¹H and ¹³C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and ³¹P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.     

Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an 'uncloneable' phenotype

Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr. These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA. A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T----G:C transitions. A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.     

13C-NMR studies of the effects of the carcinogen acetylaminofluorene on the conformation of dinucleoside monophosphate

13C-NMR spectra are obtained in aqueous solution of dinucleoside monophosphates (ApG and GpA) and of their adducts formed by the addition of the carcinogen acetylaminofluorene (AAF) to the C8 position of the guanine. The base and sugar carbons of all dimers and adducts are assigned. The task of assigning base and carbohydrate resonances was accomplished using a series of reference compounds. Significant changes in many of the carbon resonances of the adducts are observed suggesting three general conformational changes, namely: (1) chemical shift changes are noted in base carbon atom resonances as a function of temperature and adduct formation which are indicative of stacking effects; (2) large upfield shifts of the furanose C2' resonance of the guanosine-adduct indicate a shift to higher populations of the syn conformation. Other shifts of carbohydrate resonances are indicative of a change in conformation of the carbohydrate itself. (3) Large temperature effects on linewidth of several fluorine and furanose resonances indicate interconversion of various conformers in the dimer adduct.     

Fourier-transform infrared difference spectroscopy of rhodopsin and its photoproducts at low temperature

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position or intensity between rhodopsin and the photoproducts formed at low temperature (70 K), bathorhodopsin and isorhodopsin. A method has been developed to obtain infrared difference spectra between rhodopsin and bathorhodopsin, bathorhodopsin and isorhodopsin, and rhodopsin and isorhodopsin. To aid in the identification of the vibrational modes, we performed experiments on deuterated and hydrated films of native rod outer segments and rod outer segments regenerated with either retinal containing 13C at carbon 15 or 15-deuterioretinal. Our infrared measurements provide independent verification of the resonance Raman result that the retinal in bathorhodopsin is distorted all-trans. The positions of the C = N stretch in the deuterated pigment and the deuterated pigments regenerated with 11-cis-15-deuterioretinal or 11-cis-retinal containing 13C at carbon 15 are indicative that the Schiff-base linkage is protonated in rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, the C = N stretching frequency occurs at the same position in all three species. The data indicate that the protonated Schiff base has a C = N trans conformation in all three species. Finally, we present evidence that, even in these early stages of the rhodopsin photosequence, changes are occurring in the opsin and perhaps the associated lipids.     

Optical properties and base pairing of E. coli 5S RNA

The ultraviolet absorption, optical rotatory dispersion, circular dichroism, and infrared absorption spectra of renatured 5S RNA have been measured at pH 7.0 in 0.1M NaCl at 25° and used to obtain four independent estimates of the number of base pairs. These four estimations are in reaonable agreement and average values of 28 ± 4 G.C and 13 ± 4 A.U. base pairs.     

Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Structural changes in the L photointermediate of bacteriorhodopsin

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.     

Use of a rapid DNA sequencing system to demonstrate the induction of frameshift mutations by bleomycin

The rapid DNA sequencing system based on the single-stranded bacteriophage M13 and the chain-terminator method has been used to look directly for mutational alterations. A small DNA fragment that primes DNA synthesis through the N-terminal 200 base pairs of the beta-galactosidase gene was prepared, and used to detect changes in base sequence among phages that give white plaques after treatment of the host cells with bleomycin. Bleomycin treatment of E. coli in which M13 mp2 was growing gave an increase in white plaque frequency. DNA sequence analysis of phage from 7 independent mutant plaques showed them all to have a frameshift mutation.     

The influence of single base triplet changes on the stability of a pur.pur.pyr triple helix determined by affinity cleaving.

The influence of sixteen base triplet changes at a single position within a pur.pur.pyr triple helix was examined by affinity cleaving. For the 15 base pair target site studied here, G.GC, A.AT and T.AT triplets stabilize a triple helix to a greater extent than the other 13 natural triplets (pH = 7.4, 25 degrees C). Weaker interactions were detected for the C.AT, A.GC and T.CG triplets. The absence of specific, highly stabilizing interactions between third strand bases and the CG or TA base pairs demonstrates a current sequence limitation to formation of this structure. Models for the two dimensional base triplet interactions for all possible 16 natural triplets are presented.     

Localization of the retinal protonated Schiff base counterion in rhodopsin.

Semiempirical molecular orbital calculations are combined with 13C NMR chemical shifts to localize the counterion in the retinal binding site of vertebrate rhodopsin. Charge densities along the polyene chain are calculated for an 11-cis-retinylidene protonated Schiff base (11-cis-RPSB) chromophore with 1) a chloride counterion at various distances from the Schiff base nitrogen, 2) one or two chloride counterions at different positions along the retinal chain from C10 to C15 and at the Schiff base nitrogen, and 3) a carboxylate counterion out of the retinal plane near C12. Increasing the distance of the negative counterion from the Schiff base results in an enhancement of alternating negative and positive partial charge on the even- and odd-numbered carbons, respectively, when compared to the 11-cis-RPSB chloride model compound. In contrast, the observed 13C NMR data of rhodopsin exhibit downfield chemical shifts from C8 to C13 relative to the 11-cis-RPSB.Cl corresponding to a net increase of partial positive or decrease of partial negative charge at these positions (Smith, S. O., I. Palings, M. E. Miley, J. Courtin, H. de Groot, J. Lugtenburg, R. A. Mathies, and R. G. Griffin. 1990. Biochemistry. 29:8158-8164). The anomalous changes in charge density reflected in the rhodopsin NMR chemical shifts can be qualitatively modeled by placing a single negative charge above C12. The calculated fit improves when a carboxylate counterion is used to model the retinal binding site. Inclusion of water in the model does not alter the fit to the NMR data, although it is consistent with observations based on other methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and dynamics of short DNA duplexes: (dC-dG)3 and (dC-dG)4

Natural abundance 13C NMR spectra of duplexed (dC-dG)3 and (dC-dG)4 exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(1) sugar. 4M NaCl causes changes in the 13C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the anti to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

Conformation of adenosine bulge-containing deoxytridecanucleotide duplexes in solution. Extra adenosine stacks into duplex independent of flanking sequence and temperature

Structural features at the extra adenosine bulge sites in DNA duplexes have been elucidated from an NMR analysis of two-dimensional through space and through bond connectivities in the self-complementary d(C-C-G-G-A-A-T-T-C-A-C-G-G) (CAC 13-mer) and d(C-C-G-A-G-A-A-T-T-C-C-G-G) (GAG 13-mer) duplexes in aqueous solution. These studies establish that the extra adenosine stacks into the helix at all temperatures below the onset of the melting transition in solution, and the results are independent of whether the extra adenosine is flanked by cytidines (CAC 13-mer) or guanosines (GAG 13-mer). The NMR parameters establish that the extra adenosine can be accommodated into the helix with the flanking base pairs adopting a wedge-shaped orientation. The resulting perturbation extends out to the C10-G11 phosphodiester backbone adjacent to the bulge segment in both the CAC 13-mer and GAG 13-mer duplexes.     

13C-NMR of ribosyl ApApA, ApApG and ApUpG

The chemical shifts as well as the 13C-31P coupling constants of the carbon-13 nuclei in single-stranded ApApA, ApApG, and ApUpG are sensitive to sequence and temperature. ApApA and ApApG have similar properties with large shielding (up to 1.7 ppm) of many of the base carbons upon decreasing the temperature from 70 degrees C to 11 degrees C; the base carbons have smaller shielding changes in ApUpG. Large shielding and deshielding effects are observed for the 1', 3', 4' and 5'-carbons over this temperature range. Analysis of the 13C-31P couplings measured at the 4' ribose carbons show that the population of the anti rotamer about O5'-C5' varies from 98 to 75%, and is higher in ApApA and ApApG than in ApUpG. The CCOP coupling data at 2' and 4' is consistent with a blend of the -antiperiplanar/-synclinal nonclassical rotamers about the C3'-O3' bond, varying from 89/11% in ApApG to 55/45% in ApUpG. The coupling and chemical shift data support the thesis that ApUpG is stacked much less than the other two molecules. The stacked forms of all three trinucleotides is most easily interpreted by a standard A-RNA model. It is not necessary to invoke the "bulged base" hypothesis [Lee, C.-H. and Tinoco, Jr., I. (1981) Biophysical Chemistry 1, 283-294; Lankhorst, P.P., Wille, G., van Boom., J.H., Altona, C., and Haasnoot, C.A.G. (1983) Nucleic Acids Research 11, 2839-2856] to explain the contrast in 13C spectroscopic properties of ApUpG in comparison to ApApG and ApApA.     

Fourier transform infrared evidence for Schiff base alteration in the first step of the bacteriorhodopsin photocycle

The first step of the bacteriorhodopsin (bR) photocycle involves the formation of a red-shifted product, K. Fourier transform infrared difference spectra of the bR570 to K630 transition at 81 K has been measured for bR containing different isotopic substitutions at the retinal Schiff base. In the case of bacteriorhodopsin containing a deuterium substitution at the Schiff base nitrogen, carbon 15, or both, we find spectral changes in the 1600-1610- and 1570-1580-cm-1 region consistent with the hypothesis that the K630 C=N stretching mode of a protonated Schiff base is located near 1609 cm-1. A similar set of Schiff base deuterium substitutions for retinal containing a 13C at the carbon 10 position strongly supports this conclusion. This assignment of the K630 C=N stretching vibration provides evidence that the bR Schiff base proton undergoes a substantial environmental change most likely due to separation from a counterion. In addition, a correlation is found between the C=N stretching frequency and the maximum wavelength of visible absorption, suggesting that movement of a counterion relative to the Schiff base proton is the main source of absorption changes in the early stages of the photocycle. Such a movement is a key prediction of several models of proton transport and energy transduction. Evidence is also presented that one or more COOH groups are involved in the formation of the K intermediate.     

A new approach to the study of sequence-dependent properties of polynucleotides

A sequence-dependent properly of any long polynucleotide can be related to the properties of a limited number of other polynucleotides. The properties of the constituent sequence subunits need not be individually known. Only the properties of a set of linearly independent sequence combinations in polymers need be known. The number of independent sequences depends on the number of neighboring bases (or base pairs) contributing to the property and on the number of different bases (or base pairs) allowed. General formulae are derived. If only nearest neighbors contribute to a property, there are 13 linearly independent single-strand sequences with four different bases and there are 8 linearly independent double-strand sequences with two different base pairs. The study of independent sequences in polymers should be especially useful with double-stranded polynucleotides. It should be possible by this polymer approach to estimate nearest-neighbor frequencies from the circular dichroism of double strands and also to investigate the relation between solution conformation and double-strand sequence.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.