Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.     

Conformational properties of B-Z junctions in DNA.

The structural consequences of specific base sequences in DNA can exert a strong influence on the function of DNA. It has previously been reported that the presence of multiple B-Z conformational junctions in constructed DNA oligomers results in unusually enhanced electrophoretic gel mobilities of these oligomers [Winkle, S. A., & Sheardy, R. D. (1990) Biochemistry 29, 6514-6521]. In order to investigate this phenomenon further, we designed and synthesized several DNA oligomers capable of pure Z or B-Z junction formation for polyacrylamide gel electrophoresis studies. The results indicate that both pure Z-DNA and polymorphic B-Z-DNA oligomers exhibit unusual gel migratory properties. The results of gel mobility studies in the absence and presence of cobalt hexamine indicate that a B-Z junction corresponds to a stiff bend of the helix axis, with two or more conformers accessible at the junction site. This is a different bend and mechanism than that in oligo(A) tracts.     

Anomalous gel migration of DNA oligomers containing multiple conformational junctions

We have previously shown that a short 16 base pair DNA oligomer can accommodate a B-Z conformational junction [Sheardy, R. D., & Winkle, S. A. (1989) Biochemistry 28, 720-725]. Results from 1H NMR studies indicated that only three base pairs were involved in the junction and that one of these base pairs was highly distorted. Being interested in the nature of this distortion, we constructed DNA oligomers which have the potential to contain multiple B-Z junctions for polyacrylamide electrophoretic studies. We report that the mobilities displayed by these molecules through acrylamide gels in the absence and presence of cobalt suggest that these molecules run shorter than they actually are. This anomalous migration may be due to structural/dynamic properties of the DNA helix manifested by the periodic distortions of the potential B-Z junctions.     

NMR studies of ion binding to Escherichia coli tRNAPhe

The effects of magnesium, spermine, and temperature on the conformation of Escherichia coli tRNAPhe have been examined by proton and phosphorus nuclear magnetic resonance spectroscopy. In the low-field proton NMR spectra we have characterized two slowly interconverting conformations of this tRNA at low magnesium ion concentrations. The relative proportion of the conformers is ion dependent but not ion specific. Magnesium affects protons in all the stems of tRNA while spermine effects are localized near the s4U-8-A-14 and G-15-C-48 tertiary bonds. The effects seen in the proton NMR spectra are compared and correlated with those observed in the phosphorus spectra to give assignments of some of the resolved signals from the phosphate groups. The phosphorus spectra are compared with those of yeast tRNAPhe [Gorenstein, D. G., Goldfield, E. M., Chen, R., Kovar, K., & Luxon, B. A. (1981) Biochemistry 20, 2141; Salemink, P. J. M., Reijerse, E. J., Mollevanger, L., & Hilbers, C. W. (1981) Eur. J. Biochem. 115, 635], and the ion effects are discussed with reference to the magnesium and spermine sites found in the crystal structures of yeast tRNAPhe [Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, S.-H. (1977) Nucleic Acids Res. 4, 2811; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64; Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315].     

Two- and three-dimensional proton NMR studies of apo-neocarzinostatin.

Neocarzinostatin (NCS) is an antitumor protein from Streptomyces carzinostaticus that is identical in apo-protein sequence with mitomalcin (MMC) from Streptomyces malayensis. We describe the use of apo-NCS as a model system for applying combined two- and three-dimensional (2D and 3D) proton NMR spectroscopy to the structure determination of proteins (Mr greater than 10K) without isotope labeling. Strategies aimed at accurately assigning overlapped 2D cross-peaks by using semiautomated combined 2D and 3D data analysis are developed. Using this approach, we have assigned 99% of the protons, including those of the side chains, and identified about 1270 intra- and interresidue proton-proton interactions (fixed distances are not included) in apo-NCS. Comparing our results with those reported recently on 2D NMR studies of apo-NCS [Adjadj, E., Mispelter, J., Quiniou, E., Dimicoli, J.-L., Favadon, V., & Lhoste, J.-M. (1990) Eur. J. Biochem. 190, 263-271; Remerowski M. L., Glaser, S. J., Sieker, L., Samy, T. S. A., & Drobny, G. P. (1990) Biochemistry 29, 8401-8409] demonstrated advantages of proton 3D NMR spectroscopy in protein spectral assignments. We are able to obtain more complete proton resonance and secondary structural assignments and find several misassignments in the earlier report. Strategies utilized in this work should be useful for developing automation procedures for spectral assignments.     

Preparation and characterization of spin-labeled oligonucleotides for DNA hybridization.

Sequence-specific spin-labeled oligodeoxynucleotides with conformation-sensitive electron paramagnetic resonance (EPR) signals are synthesized and examined as solution-phase nucleic acid hybridization probes. Either a proxyl or tempo ring linked to the C(5) position of deoxyuridine (dU) by a nonrigid two-atom methylamino tether is incorporated within 15-mers by phosphotriester chemistry yielding stable spin-labeled probes with distinctive EPR specific activity (AEPR) values. The AEPR is greater for a proxyl-labeled than for a tempo-labeled probe and is consistent with EPR data of enzymatically labeled 26-mers [Bobst, A. M., Pauly, G. T., Keyes, R. S., and Bobst, E. V. (1988) FEBS Lett. 228, 33-36], after normalizing for percent labeling. The spectral characteristics of the free probes and the probe/target complexes are similar to those of enzymatically spin-labeled nucleic acids containing a different nonrigid two-atom-tethered spin label [Bobst, A. M., Kao, S.-C., Toppin, R. C., Ireland, J. C., and Thomas, I. E. (1984) J. Mol. Biol. 173, 63-70]. The presence of target DNA is detected in solution by EPR spectroscopy and the assay is based on the characteristic line-shape change associated with hybridization. The EPR spectra of free and bound probe reflect little interference from changes in global dynamics of the probe, and the line-shape change upon complexation results primarily from a change in local base dynamics. The presence or absence of hybridization can be detected in a loop-gap resonator with about 1 pmol of spin-labeled 15-mer within minutes.     

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions.

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(A ACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The DNA-RNA junction residues exhibit quite different COSY, chemical shift, and NOE behavior, but these effects do not appear to propagate into the DNA or RNA segments. The circular dichroism spectra of these d-r-d chimeras also display a mixture of characteristic A-type and B-type absorption bands. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but our results differ somewhat from previous theoretical model studies.     

Design of a helix-bundle cross-link: NMR and UV-visible spectroscopic analyses and molecular modeling of ring-oxidized retinals

In order to generate potential chemical cross-links for studying the chromophore binding site of bacteriorhodopsin and related helix-bundle proteins, MnO2 was used to oxidize all-trans-retinal's ring moiety. The structures and solution conformations of three ring-oxidized retinal analogues have been determined by using UV-visible absorption and 1H and 13C NMR spectroscopies, primarily with regard to (i) the introduction of a functional group at the ring end of the chromophore, (ii) the retention of the all-trans geometry of the polyenal side chain, and (iii) the torsional angle of the ring-polyenal bond. Analyses of their UV-visible absorption spectral parameters (lambda max, epsilon max, and vibrational fine structure) and NMR spectral parameters (1H-1H coupling constants, 1H and 13C NMR chemical shifts, and 1H homonuclear Overhauser effects) indicated the 4-oxo and the 2,3-dehydro-4-oxo derivatives both possess the twisted 6-s-cis conformation adopted by most six-membered ring analogues of retinal in solution or crystal. However, the alpha-dioxocyclopentenyl analogue exists in solution predominantly (70-80%) as the planar 6-s-trans conformer, similar to violerythrine chromophore analogues. In order to identify the minor solution forms, molecular modeling and geometry optimizations using the semiempirical molecular orbital method AM1 defined two additional symmetry-related minima at +/- 30-40 degrees in its C6-C7 torsional energy profile. Because the chromophores of bacterio- and halorhodopsins and sensory rhodopsins are bound as the 6-s-trans conformer [Harbison, G.S., Smith, S.O., Pardoen, J.A., Courtin, J.M.L., Lugtenburg, J., Herzfeld, J., Mathies, R.A., & Griffin, R.G. (1985) Biochemistry 24, 6955-6962; Baselt, D.R., Fodor, S.P.A., van der Steen, R., Lugtenburg, J., Bogomolni, R.A., & Mathies, R.A. (1989) Biophys. J. 55, 193-196], we suggest that the cyclopentenyl analogue's alpha-diketo function may be favorably positioned within the binding pocket and sufficiently reactive toward nucleophilic attack to cross-link an arginine located in or near the ring end of the chromophore cavity: Arg134 according to the current model of bacteriorhodopsin's tertiary structure [Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckmann, E., & Downing, K.H. (1990) J. Mol. Biol. 213, 899-929] or Arg82 as postulated from an alternate model constructed primarily to accommodate the external point charge contribution to bacteriorhodopsin's opsin shift.     

Variation of nonexchangeable proton resonance chemical shifts as a probe of aberrant base pair formation in DNA

Variation of nonexchangeable proton resonance chemical shifts for deoxycytidine and deoxy-adenosine as a function of protonation and imino tautomer formation has been determined. Protonation induces downfield shifts of proton resonances whereas formation of the rare imino tautomer induces upfield shifts. Titration curves are constructed on the basis of spectrophotometrically determined pK values. Excellent fit is obtained between theoretical titration curves and experimental data, which indicates that chemical shifts of base protons may be used to quantitatively determine the relative concentrations of either rare imino tautomeric conformations or protonated base forms. These data may be utilized as an aid in the elucidation of the nature of hydrogen bonding between mismatched base pairs in DNA oligomers containing cytosine or adenine residues. These data, in conjunction with the oligonucleotide study of Patel et al. [Patel, D. J., Kozlowski, S.A., Ikuta, S., & Itakura, K. (1984) Biochemistry 23, 3218-3226], have been used to rigorously argue the existence of a "protonated" adenine residue in the A-C mismatch. This structure allows reconciliation of the NMR solution data with crystallographic data [Hunter, W.N., Brown, T., Anand, N.N., & Kennard, O. (1986) Nature (London) 320, 552-555], which support the protonated base pair.     

A.T and C.C+ base pairs can form simultaneously in a novel multistranded DNA complex

Previous experiments have established that in certain synthetic oligomeric DNA sequences, including mixtures of d(AACC)5 with d(CCTT)5, adenine-thymine (A.T) base pairs form to the exclusion of neighboring protonated cytosine-cytosine (C.C+) base pairs [Edwards, E., Ratliff, R., & Gray, D. (1988) Biochemistry 27, 5166-5174]. In the present work, circular dichroism and other measurements were used to study DNA oligomers that represented two additional classes with respect to the formation of A.T and/or C.C+ base pairs. (1) One class included two sets of repeating pentameric DNA sequences, d(CCAAT)3-6 and d(AATCC)4,5. For both of these sets of oligomers, an increase in the magnitude of the long-wavelength positive CD band centered at about 280 nm occurred as the pH was lowered from 7 to 5 at 0.1 and 0.5 M Na+, indicating that C.C+ base pairs formed. Even though it may have been possible for these oligomers to form duplexes with two antiparallel A.T base pairs per pentamer, no A.T base pairing was detected by monitoring the CD changes at 250 nm. Thus, spectral data showed that as few as 40% C.C+ base pairs were stable in two sets of oligomers in which A.T base pairs did not form adjacent to, or in place of, C.C+ base pairs. (2) Another class of oligomer was represented by d(C4A4T4C4), which was studied by CD, HPLC, and centrifugation experiments. We confirmed previous work that this sequence was able to form both types of base pairs as the pH and temperature were lowered [Gray, D., Cui, T., & Ratliff, R. (1984) Nucleic Acids Res. 12, 7565-7580].(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure

In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.     

Protein-bound conformation of a specific inhibitor against Candida albicans myristoyl-CoA:protein N-myristoyltransferase in the ternary complex with CaNmt and myristoyl-CoA by transferred NOE measurements.

Transferred nuclear Overhauser enhancement (trNOE) experiments have been performed to study the bioactive conformation(s) of Ro09-3472/000 derivatives in the ternary complex with Candida albicans myristoyl-CoA: protein N-myristoyltransferase (CaNmt) and myristoylCoA (MyrCoA). A critical step in the trNOE study is to identify 'true' trNOEs in the spectra. Nonspecific binding of ligands to target proteins and/or spin diffusion effects can give rise to 'false' trNOEs, which may lead to an incorrect conclusion if used to derive bound conformations. In this study for all ligands the observed trNOEs arose from specific binding interactions with the active site of CaNmt. This was shown by displacing the ligand with the known tightly binding active-site inhibitor 1 [Devadas, B., Zupec, M.E., Freeman, S.K., Brown, D.L., Nagarajan, S., Sikorski, J.A., McWherter, C.A., Getman, D. P. & Gordon, J.I. (1995) J. Med. Chem. 38, 1837-1840] and measuring the resonance linewidths in the NMR spectrum before and after addition of the competitive inhibitor. The compounds were also tested for nonspecific protein binding with bovine serum albumin (BSA) using the same METHOD: Of the six compounds tested, Ro09-3700/001 (racemate) and its optically pure enantiomers, Ro09-4764/001(S) and Ro09-4765/001(R), showed both specific binding to CaNmt and no interaction with BSA. The NMR data of these molecules in the ternary complex with CaNmt/MyrCoA could thus be used for a detailed structural analysis. Thereby, the conformation of the bound ligand was obtained from a conformational search using the observed trNOEs as a selection filter. The NMR-determined conformations are in good agreement with the recently solved CaNmt-bound X-ray structures of two similar Ro09-3472/000 derivatives.     

The position of arginine 124 controls the rate of iron release from the N-lobe of human serum transferrin. A structural study

Human serum transferrin (hTF) is a bilobal iron-binding and transport protein that carries iron in the blood stream for delivery to cells by a pH-dependent mechanism. Two iron atoms are held tightly in two deep clefts by coordination to four amino acid residues in each cleft (two tyrosines, a histidine, and an aspartic acid) and two oxygen atoms from the "synergistic" carbonate anion. Other residues in the binding pocket, not directly coordinated to iron, also play critical roles in iron uptake and release through hydrogen bonding to the liganding residues. The original crystal structures of the iron-loaded N-lobe of hTF (pH 5.75 and 6.2) revealed that the synergistic carbonate is stabilized by interaction with Arg-124 and that both the arginine and the carbonate adopt two conformations (MacGillivray, R. T. A., Moore, S. A., Chen, J., Anderson, B. F., Baker, H., Luo, Y. G., Bewley, M., Smith, C. A., Murphy, M. E., Wang, Y., Mason, A. B., Woodworth, R. C., Brayer, G. D., and Baker, E. N. (1998) Biochemistry 37, 7919-7928). In the present study, we show that the two conformations are also found for a structure at pH 7.7, indicating that this finding was not strictly a function of pH. We also provide structures for two single point mutants (Y45E and L66W) designed to force Arg-124 to adopt each of the previously observed conformations. The structures of each mutant show that this goal was accomplished, and functional studies confirm the hypothesis that access to the synergistic anion dictates the rate of iron release. These studies highlight the importance of the arginine/carbonate movement in the mechanism of iron release in the N-lobe of hTF. Access to the carbonate via a water channel allows entry of protons and anions, enabling the attack on the iron.     

G protein-bound conformation of mastoparan-X, a receptor-mimetic peptide.

Mastoparans are a family of 14-residue peptide toxins from wasp venom which have been proposed to stimulate secretion from a variety of cells, by directly activating GTP-binding regulatory proteins (G proteins). In vitro studies have shown that mastoparans activate G proteins by a mechanism remarkably similar to that used by agonist-bound receptors (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. M. (1988) J. Biol. Chem. 263, 6491-6494). Here, we report the conformation of mastoparan-X (INWKGIAAMAKKLL-NH2) when it is bound to the alpha subunits of recombinant G(i) and G(o), derived from an analysis of transferred nuclear Overhauser effects in a two-dimensional 1H NMR spectrum of mastoparan-X obtained in the presence of these G proteins. Restrained molecular dynamic simulations with NMR-derived distance constraints were used to determine conformations consistent with NMR data. The G(i)- and G(o)-bound conformations of mastoparan-X are very similar, and in both cases, a major part of the molecule adopts an amphiphilic alpha-helical conformation. The lysine residues are known to be crucial for activity, and it is thus likely that at least the polar face of the amphiphilic helix is in contact with the G proteins. These conclusions should be useful in the design of potent and selective analogs of mastoparan and in the development of models for receptor-G protein interaction.     

The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain

The progressive aggregation and deposition of amyloid beta-protein (Abeta) in brain regions subserving memory and cognition is an early and invariant feature of Alzheimer's disease, the most common cause of cognitive failure in aged humans. Inhibiting Abeta aggregation is therapeutically attractive because this process is believed to be an exclusively pathological event. Whereas many studies have examined the aggregation of synthetic Abeta peptides under nonphysiological conditions and concentrations, we have detected and characterized the oligomerization of naturally secreted Abeta at nanomolar levels in cultures of APP-expressing CHO cells [Podlisny, M. B., Ostaszewski, B. L., Squazzo, S. L., Koo, E. H., Rydell, R. E., Teplow, D. B., and Selkoe, D. J. (1995) J. Biol. Chem. 270, 9564-9570 (1); Podlisny, M. B., Walsh, D. M., Amarante, P., Ostaszewski, B. L., Stimson, E. R., Maggio, J. E., Teplow, D. B., and Selkoe, D. J. (1998) Biochemistry 37, 3602-3611 (2)]. To determine whether similar species occur in vivo, we probed samples of human cerebrospinal fluid (CSF) and detected SDS-stable dimers of Abeta in some subjects. Incubation of CSF or of CHO conditioned medium at 37 degrees C did not lead to new oligomer formation. This inability to induce oligomers extracellularly as well as the detection of oligomers in cell medium very early during the course of pulse-chase experiments suggested that natural Abeta oligomers might first form intracellularly. We therefore searched for and detected intracellular Abeta oligomers, principally dimers, in primary human neurons and in neuronal and nonneural cell lines. These dimers arose intracellularly rather than being derived from the medium by reuptake. The dimers were particularly detectable in neural cells: the ratio of intracellular to extracellular oligomers was much higher in brain-derived than nonbrain cells. We conclude that the pathogenically critical process of Abeta oligomerization begins intraneuronally.     

Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers

Genetic expansion diseases have been linked to the properties of triplet repeat DNA sequences during replication. The most common triplet repeats associated with such diseases are CAG, CCG, CGG, and CTG. It has been suggested that gene expansion occurs as a result of hairpin formation of long stretches of these sequences on the leading daughter strand synthesized during DNA replication [Gellibolian, R., Bacolla, A., and Wells, R. D. (1997) J. Biol. Chem. 272, 16793-7]. To test the biophysical basis for this model, oligonucleotides of general sequence (CNG)(n), where N = A, C, G, or T and n = 4, 5, 10, 15, or 25, were synthesized and characterized by circular dichroism (CD) spectropolarimetry, optical melting studies, and differential scanning calorimetry (DSC). The goal of these studies was to evaluate the influence of sequence context and oligomer length on their secondary structures and stabilities. The results indicate that all single oligomers, even those as short as 12 nucleotides, form stable hairpin structures at 25 degrees C. Such hairpins are characterized by the presence of N:N mismatched base pairs sandwiched between G:C base pairs in the stems and loops of three to four unpaired bases. Thermodynamic analysis of these structures reveals that their stabilities are influenced by both the sequence of the particular oligomer and its length. Specifically, the stability order of CGG > CTG > CAG > CCG was observed. In addition, longer oligomers were found to be more stable than shorter oligomers of the same sequence. However, a stability plateau above 45 nucleotides suggests that the length dependence reaches a maximum value where the stability of the G:C base pairs can no longer compensate the instability of the N:N mismatches in the stems of the hairpins. The results are discussed in terms of the above model proposed for gene expansion.     

Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).     

Sequence specific conformation of a DNA decamer containing an adenine tract studied in solution by H-NMR spectroscopy

The decanucleotide duplex d(AAAACGTTTT)2 and a variety of phase-sensitive two-dimensional (2D) NMR experiments have been used to investigate the solution conformation of an adenine-tract and its junction with another DNA sequence. 2D nuclear Overhauser effect data confirm that the oligonucleotide has a general B-type DNA morphology but an array of unusual correlations implies that the adenine tract and the 5'-ApC junction have conformations more compatible with the modified X-ray structures recently reported for DNAs of similar sequence (Nelson, H.C.M., Finch, J.T., Luisi, B.F. and Klug, A. (1987) Nature 330, 221-226). The pattern and magnitude of interstrand NOEs from the adenine H2s to the sugar H1's of the complementary base to the 5'-neighbouring residue indicate that the A-T basepairs are highly propeller twisted and that the minor groove is narrowed, showing its greatest compression at the 3'-end of the tract at the 5'-ApC step. Quantifying spin-coupling interactions within the deoxyribose rings by analysing both 1D and high-resolution 2D DQF-COSY data reveals that the conformation of the purines is predominantly C2'-endo, with the pseudorotation phase angle P lying in the range 140-180 degrees. For the pyrimidines, however, there are distortions away from this standard B-type geometry with the data being best described by P values lying in the range 90-130 degrees (i.e., O4'-endo, C1'-exo). The sugar puckers of A1, T9 and T10 are dynamically distorted no doubt as a consequence of their positions at, or close to, the ends of the duplex. Thus the conformation of the adenine and thymine sugars within the oligo(dA) and oligo(dT) strands are different with an abrupt change in sugar puckering occurring at the 5'-ApC (5'-GpT) step. Peculiar chemical shifts values for A4H2, T7CH3 and sugar C5 H1', H2' and H2", together with a number of interresidue NOEs with unusual intensities, imply that there are also substantial modifications to basepair stacking interactions at this step. Taken as a whole, our data are consistent with the view that the conformational dislocation at the 5'-ApC dinucleotide results from a combination of slide and roll manoeuvres and that the junction between the AAAA and CG sequences is a potential nucleation site for DNA bending.     

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate.

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559).(ABSTRACT TRUNCATED AT 250 WORDS)