Biosynthetic incorporation of 15N and 13C for assignment and interpretation of nuclear magnetic resonance spectra of proteins.
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Amino Acid Sequence↗
Biomedical subjects
L P McIntosh
Publications and source records attributed to L P McIntosh.
Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance.
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Escherichia coli↗
Use of 13C and 15N isotope labels for proton nuclear magnetic resonance and nuclear Overhauser effect. Structural and dynamic studies of larger proteins and nucleic acids.
This article reviews methods based on direct observation of proton NMR in macromolecules containing 13C or 15N labels. The resonances and Overhauser effects of protons attached to the labels can be edited or filtered from the remaining overlapping resonances. This leads to simplification of the spectra when labels are incorporated selectively. In 2D and related methods the label's chemical shift provides a second dimension which is useful for spectral differentiation and identification. The methods are useful for larger proteins and we describe our progress on studies of T4 lysozyme, mass 18.7 kD, in which we have already identified a large number of resonances.
Carbon Isotopes↗
Vacuum UV circular dichroism is diagnostic for the left-handed Z form of poly [d(A-C).d(G-T)] and other polydeoxynucleotides.
Circular dichroism spectra are extended into the vacuum UV to about 178 nm for four polydeoxynucleotides of various sequences capable of assuming the left-handed Z form. It is found that each of these polymers, including those with brominated bases and those with the four different bases, have a characteristic negative feature at short wavelengths when in the Z form. In contrast, the B form only has a positive band between 180 and 200 nm. Furthermore, a blue shift of the short wavelength crossover is diagnostic of the B- to Z-form transition for all polymers studied so far. These results confirm that poly[d(A-C).d(G-T)] can assume the Z form in solution at low concentration.
Circular Dichroism↗
Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins.
A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on 15N (or 13C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-15N (or 13C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from 15N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-15N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl[15N]alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.
Deuterium↗
Proton NMR and NOE structural and dynamic studies of larger proteins and nucleic acids aided by isotope labels: T4 lysozyme.
This article reviews methods based on direct observation of proton NMR in macromolecules containing 13C or 15N labels. The resonances and Overhauser effects of protons attached to the labels can be edited or filtered from the remaining overlapping resonances. This leads to simplification of the spectra when labels are incorporated selectively. In 2D and related methods the label's chemical shift provides a second dimension which is useful for spectral differentiation and identification. The methods are useful for larger proteins and we describe our progress on studies of T4 lysozyme, mass 18.7 kD, in which we have already identified a large number of resonances.
Carbon Isotopes↗
1H nuclear magnetic resonance study of the dynamic properties of the B and Z-forms of poly[d(A-br5C).d(G-T)].
Poly[d(A-br5C).d(G-T)], a synthetic polynucleotide with a 50% A-T base composition, undergoes a reversible, highly co-operative transition between the right-handed B and left-handed Z conformations. The latter is stabilized at both elevated temperature and ionic strength. The B and Z-forms of poly[d(A-br5C).d(G-T)] coexist in 4.6 M-NaCl at 45 degrees C. Due to slow exchange, two sets of Tim and Gim resonances are observed and can be assigned to the B and Z conformations (the chemical shifts are, respectively, Tim = 13.4, 14.1 p.p.m. (parts/million); and Gim = 11.9, 12.4 p.p.m.). Measurements of the 1H spin-lattice (R1) and spin-spin (R2) relaxation rates of the exchangeable thymine (Tim) and guanine (Gim) imino protons have been used to probe the internal dynamics of the B and Z-forms of poly[d(A-br5C).d(G-T)] and the mechanism of the B-Z transition. The proton exchange behavior in the B and Z conformations is quite different. At elevated temperature, R1 for both Tim and Gim in the B conformation is dominated by exchange with the solvent, with Tim exchanging more rapidly than Gim. This demonstrates that exchange involves the opening of single base-pairs and that neighboring A-T and G-br5C base-pairs exchange independently of each other. B-form poly[d(A-br5C).d(G-T)] is unusual in that there is an acceleration of the Tim exchange rate with increasing NaCl concentration. Conversion to the Z-form by addition of 4.5 M-NaCl dramatically reduces both the Tim and Gim exchange rates (estimated to be less than 2 s-1 at 70 degrees C). Thus, the G-br5C base-pair and, in particular, the A-T base-pair are stabilized in the Z conformation. By measuring relaxation rates at 45 to 50 degrees C where the B and Z-forms are in equilibrium, we find that the B-Z interconversion rates are less than two per second. In the B conformation at 25 degrees C, the dipolar contributions to the imino proton relaxation rates are about one-third of those expected on the basis of a rigid rod model for 65 base-pair fragments, a difference we assign to large amplitude (30 degrees high frequency (less than 100 ns) out-of-plane motions of the bases. Conversion to the Z conformation has little effect on the dipolar contributions to relaxation, i.e. on the internal motions.(ABSTRACT TRUNCATED AT 400 WORDS)
Magnetic Resonance Spectroscopy↗
Synthesis and characterization of poly[d(G-z5C)]. B-Z transition and inhibition of DNA methylase.
Deoxy-5-azacytidine 5'-triphosphate was synthesized and used as a substrate for the enzymatic synthesis of the polynucleotide poly[d(G-z5C)]. Whereas the triphosphate decomposes in solution, the azacytosine analogue incorporated into DNA is stable under conditions preserving the double-helical structure. Poly[d(G-z5C)] undergoes the transition to the left-handed Z conformation at salt (NaCl and MgCl2) concentrations approximately 30% higher than those required for unsubstituted poly[d(G-C)]. However, the incorporation of azacytidine potentiates the formation at room temperature of the Z helix stabilized by the transition metal Mn2+; in the case of poly[d(G-C)], a heating step is required. The spectral properties of the two polymers in the B and Z forms are similar. Both left-handed forms are recognized by anti-Z DNA immunoglobulins, indicating that the DNAs bear common antigenic features. Poly[d(G-z5C)] is not a substrate for the DNA cytosine 5-methyltransferase from human placenta. It is a potent inhibitor of the enzyme when tested in a competitive binding assay. These results are compatible with a very strong, possibly covalent, mode of interaction between methyltransferases and DNA containing 5-azacytosine.
Azacitidine↗
Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences.
The relative immunogenicities of the poly[d(G-C)] and poly[d(A-C).d(G-T)] families of helices have been determined. The specificities of the resultant immunoglobulins have been characterized for recognition of different synthetic and natural left-handed sequences and conformations. Certain modifications of poly[d(G-C)] in the sugar-phosphate backbone and cytosine C-5 potentiate the right(R)-to-left(L) (B----Z) transition under physiological conditions. The resulting polynucleotides, poly[d(G-SC)], poly[d(G-io5C)], poly[d(G-br5C)] and poly[d(G-m5C)], are also highly immunogenic. In contrast, DNAs incapable of assuming the left-handed conformation under physiological salt concentrations are weakly or non-immunogenic. These include unmodified poly[d(G-C)] as well as members of the poly[d(A-C).d(G-T)] family of sequences bearing pyrimidine C-5 substitutions (methyl, bromo, iodo). These polynucleotides undergo the R----L isomerization under more stringent ionic and thermal conditions. The specificities of purified polyclonal and monoclonal anti-Z DNA immunoglobulins (IgG) were measured by binding to radiolabeled polynucleotides, by electrophoretic analysis of IgG bound to covalent closed circular DNAs, and by immunofluorescent staining of polytene chromosomes. The salt-induced left-handed forms of poly[d(G-C)] and its derivatives (including the cytidine C-5 methyl, bromo, iodo, and N-5 aza substituted polynucleotides) and of the modified poly[d(A-C).d(G-T)] polymers are bound to varying degrees by different antibodies. The patterns of substrate recognition demonstrate the existence of several antigenic domains in left-handed DNAs, including the helix convex surface and the sugar-phosphate backbone. Substitutions in these regions can produce enhancing (required substitutions), neutral, or inhibitory effects on subsequent IgG binding. Additionally, certain modifications of either the convex surface of Z DNA at the C-5 position of cytidine (i.e. a methyl group) or of the backbone (i.e. phosphorothioate substitution) can lead to polymorphic left-handed conformations that are compatible with antibody binding when present individually but not in combination. The recognition patterns exhibited with DNA substrates from the two DNA families indicate that some, but not all, IgGs show specificity for different nucleotide sequences. The anti-Z DNA IgGs were used to probe for specific left-handed Z DNA determinants on plasmid (e.g. pBR322) or viral (e.g. simian virus 40 (SV40] DNAs and on the acid-fixed polytene chromosomes of dipteran larvae.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Interactions of anti-poly[d(G-br5C)] IgG with synthetic, viral and cellular Z DNAs.
Enzymatically synthesized poly[d(G-br5C)] was used to prepare specific polyclonal and monoclonal anti-Z DNA IgGs. The binding specificities of these antibodies were characterized using left-handed polynucleotides with the sequences d(G-x5C)n and d(A-x5C)n.d(G-T)n (mean = aza, methyl, bromo, or iodo). Polyclonal anti-poly[d(G-br5C)] IgG binds the convex surface of the Z helix as evidenced by the strong requirement for a methyl or halogen group at the C5 position of cytosine. Little or no anti-poly[d(G-br5C)] IgG binding occurs to left-handed DNAs carrying a phosphorothioate substitution in the dGpdC bond or an N-5 aza substitution in the cytosine ring. Anti-poly[d(G-br5C)] IgG can stabilize transient Z DNA structures in both polymer families, thereby displacing the equilibrium in solution between the right-and left-handed DNA conformations. Anti-poly[d(G-br5C)] IgG binding sites are found in all tested covalently closed circular natural DNAs (Form I) at their extracted negative superhelical densities, but not in any of the corresponding relaxed Form II or linear Form III DNAs. Binding of anti-poly[d(G-br5-C)] IgG leads to a reduction in the electrophoretic mobility of Form I DNA (e.g. SV40, phi X174, or pBR322) and to the formation of dimers comprised of the bivalent antibody and two supercoiled Form I DNA molecules. The dimers are converted to monomers by DTT treatment. The formation of IgG-DNA complexes is dependent on external conditions (ionic strength, temperature), the properties of the DNA (torsional stress, sequence), and the immunoglobulin (specificity, valency, and concentration). Higher order oligomeric species, indicative of two or more left-handed segments per DNA molecule are formed in reactions of anti-poly[d(G-br5C)] IgG with M13 RF I DNA but not with SV40, pBR322, or phi X174 DNAs. However, oligomers of the latter are generated with other anti-Z DNA IgGs having a broader spectrum of anti-Z DNA reactivity. Conditions which destabilize natural Z sequences in deproteinized supercoiled genomes are: monovalent salt concentrations at or above the 'physiological' range, high temperature, and topological relaxation with DNA gyrase (in the absence of ATP) or with type I topoisomerases. DNA gyrase (plus ATP) catalyses an increase in DNA negative superhelical density which leads to greater anti-Z DNA IgG binding, indicating the formation of additional left-handed regions. Polytene chromosomes of insect larvae bind anti-poly[d(G-br5C)] IgG specifically and stably at Z DNA sites. The distribution of this IgG binding differs in certain regions from that displayed by anti-Z DNA IgG probes with other sequence specificities.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Left-handed DNA: from synthetic polymers to chromosomes.
The interconversions between right-handed (R) and left-handed (L) helical conformations of DNA have been assessed by spectroscopic, electrophoretic, immunochemical, and enzymatic techniques. We have screened salt and solvent conditions which facilitate these transitions, as well as certain chemical modifications of the bases and backbone of defined synthetic polynucleotides. These include major and minor groove substituents as well as phosphorothioate analogues of selected phosphodiester bonds. We have established: R-L transitions in poly[d(G-C)] with iodo, bromo, methyl, and aza substitutions at the C5 position of cytosine, or phosphorothioate modification of the dGpC linkage. R-L transitions in the [d(A-C).d(G-T)]n sequence family using polymers modified as in the case of poly[d(G-C)]. The isomerizations are highly salt and temperature dependent. a possible L form of poly[d(A-T)] substituted with 2-amino adenine. the immunogenicities of constitutive and facultative Z-DNAs. the recognition specificities of different anti-Z-DNA IgGs for the spectrum of available polynucleotide probes. Some IgGs are sequence-specific. stabilization by IgG of otherwise transient left-handed conformations. anti-Z-DNA IgG binding to acid-fixed polytene chromosomes from the Diptera Drosophila, Chironomus, and Glyptotendipes. Laser scanning microscopy shows a maximal binding of 1 IgG per 3000-15,000 basepairs in acid fixed preparations. anti-Z-DNA IgG binding to negatively supercoiled plasmid, viral, phage, and recombinant closed circular DNAs. transcription from Z and Z* (associated) left-handed templates. From these and other results we propose that Z*-DNA may have important structural-functional roles in the cell.
Animals↗
Generation of left-handed Z-DNA in solution and visualization in polytene chromosomes by immunofluorescence.
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Animals↗
Mn2+ and other transition metals at low concentration induce the right-to-left helical transformation of poly[d(G-C)].
The effects of the first-row transition metal ions on the right(B)- to left(Z)-handed helical transition of poly[d(G-C)] have been determined. The Z conformation is induced by MnCl2 at submillimolar concentrations. The forward reaction has a very large activation energy (440 kJ/mol) so that a facile conversion occurs only at temperatures above 45 degrees C. However, the left-handed form remains stable upon cooling. The addition of ethanol (20% v/v) eliminates the requirement for elevated temperature. The transition is highly co-operative and is accompanied by spectral changes (absorption, circular dichroism) characteristic for the B----Z conformational transition. NiCl2 and CoCl2 also induce the B----Z transition in poly[d(G-C)] but the activation energies and thus the temperature requirements for the forward reaction are lower than those observed with MnCl2. The left-handed DNA formed in the presence of Mn2+ is similar to 'Z DNA' previously described in Mg2+-EtOH (van de Sande and Jovin , 1982): (a) it readily sediments out of solution at low speed as a consequence of intermolecular association which, however, is not accompanied by turbidity; and (b) it supports the binding of ethidium bromide although this drug interacts preferentially with the B form of DNA. With Ni2+, the B----Z isomerization step can be separated from the subsequent specific Z----Z* association. Mn2+, Ni2+, and Co2+ also promote the B----Z transition of poly[d(G-m5C)] at substoichiometric concentrations with respect to DNA nucleotide.
Calorimetry↗
Left-handed helical conformation of poly[d(A-m5C).d(G-T)].
Poly[d(G-C)] serves as the prototype for the right-to-left (B to Z) transition in he helical sense of DNA, both in solution and inthe crystal form. However, the question remains as to which other synthetic and natural DNAs have the potential to adopt left-handed conformations. One logical candidate is the canonical alternating purine-pyrimidine sequence d(A-C)n.d(G-T)n which seems to be widely disseminated in eukaryotic genomes. Our approach to this question is based on the enzymatic synthesis of poly[d(A-C).d(G-U)] derivatives with systematic methyl and halogen substitutions in the C-5 position of the pyrimidines C and U. Such modifications in poly[d(G-C)] have previously been shown to potentiate the B to Z transition. Here we report a highly cooperative, reversible, salt- and temperature-dependent transition for poly[d(A-m5C).d(G-T)], a repeat of the d(A-m5C) sequence which may occur in natural DNA. Spectroscopic studies and the demonstrated ability to bind anti-Z-DNA antibodies suggest that the new helical conformation is left-handed and shares structural features with known Z-DNA. However, a novel property, not exhibited by poly[d(G-C)], is the profound temperature dependence of the conformational equilibrium.
Circular Dichroism↗