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A G Redfield

Publications and source records attributed to A G Redfield.

At least 37 records · Page 2Linked to original sources
PubMed1988

Structure of a pre-mRNA branch point/3' splice site region.

We have analyzed the solution structure of RNA containing the branch point/3' splice site region of the first intervening sequence (IVS1) of human beta-globin pre-mRNA by nuclease mapping and NMR. Nuclease mapping indicates that there are two distinct structural domains: one contains the branch point region, and the other includes the 3' splice site and second exon sequences. In the branch point domain, the adenosine at which the RNA branch forms appears to be in the loop of a stem/loop structure. The branch point structure does not appear to interact with other parts of the RNA, since its unique nuclease digestion pattern is conserved among transcripts containing the entire intron or only the branch point region. This is confirmed by a comparison of the NMR spectra of two RNA transcripts; a distinct set of resonances appears in the spectra of the RNA containing only branch sequences or including 3' splice site/exon 2 sequences. NMR studies further show that the 3' splice site/exon 2 domain has a lower melting temperature than the branch point domain, suggesting that the two regions are distinct dynamically as well as structurally. Nuclease mapping studies of adenovirus major late IVS1 indicate that this RNA has structural features in common with the human beta-globin transcript.

Base Sequence↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1986

NMR study of isoleucine transfer RNA from Thermus thermophilus.

An NMR and nuclear Overhauser effect (NOE) analysis of Thermus thermophilus tRNAIle1a is presented. This species contains modifications including s2T54 and s4U8 [Horie, N., Hara-Yokoyama, M., Yokoyama, S., Watanabe, K., Kuchino, Y., Nishimura, S., & Miyazawa, T. (1985) Biochemistry 24, 5711-5715]. All the expected secondary and reverse Hoogsteen AU pairs were identified, with one possible exception. The general geometry of the T psi C loop is the same as the Escherichia coli species, and there is NOE evidence for an A9-UA12 triple. Preliminary measurements of solvent exchange rates of internally hydrogen-bonded bases suggest that this tRNA is more stable than previously studied E. coli and yeast tRNAs.

Base Sequence↗
PubMed1986

Isotope-detected 1H NMR studies of proteins: a general strategy for editing interproton nuclear Overhauser effects by heteronuclear decoupling, with application to phage lambda repressor.

A strategy for editing interproton nuclear Overhauser effects (NOEs) in proteins is proposed and illustrated. Selective incorporation of 13C- (or 15N)-labeled amino acids into a protein permits NOEs involving the labeled residues to be identified by heteronuclear difference decoupling. Such heteronuclear editing simplifies the NOE difference spectrum and avoids ambiguities due to spin diffusion. Isotope-detected 1H NMR thus opens to study proteins too large for conventional one- and two-dimensional NMR methods (20-75 kDa). We have applied this strategy to the N-terminal domain of phage lambda repressor, a protein of dimer molecular mass 23 kDa. A tertiary NOE from an internal aromatic ring (Phe-51) to a beta-13C-labeled alanine residue (Ala-62) is demonstrated.

DNA-Binding Proteins↗
PubMed1985

Nuclear magnetic resonance observation of the triple interaction between A9 and AU12 in yeast tRNAPhe.

The nuclear Overhauser effect (NOE) was used to identify one of the amino proton resonances of base A23 in interaction with A9. These bases form a triple with U12 in the D stem of yeast tRNAPhe. The identification was verified by finding an NOE from this amino proton to the C8 proton of A9, as determined by comparisons of NOE's in a native and a C8-deuterated sample.

Magnetic Resonance Spectroscopy↗
PubMed1985

Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme.

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.

Amides↗
PubMed1985

Kinetics of proton exchange of phosphatidylethanolamine in phospholipid vesicles.

The rate of proton exchange of the amino protons of phosphatidylethanolamine (PE) in sonicated mixed phospholipid vesicles has been determined by NMR spectroscopy. The rate of exchange increases with increasing pH and phosphate concentration. In the absence of buffer the dominant exchange process is an intrasurface reaction in which NH2 groups react via water with NH3+ groups on the outer surface. Addition of cholesterol reduces the rate constant for intrasurface exchange. The experiments are evidence that such reactions could be dominant in proton transport in and to membrane surfaces.

Hydrogen-Ion Concentration↗
PubMed1984

Nitrogen-15-labeled yeast tRNAPhe: double and two-dimensional heteronuclear NMR of guanosine and uracil ring NH groups.

5N1-Labeled hypoxanthine and 1,3-15N-labeled uracil were synthesized chemically and used to prepare labeled yeast tRNAPhe biosynthetically. Maps (500 MHz) of 15N chemical shift vs. proton chemical shift were obtained, for each ring NH group, by means of INDOR (difference heterodecoupling) and also by means of a proton-observe two-dimensional method involving coherences of forbidden resonances of the NH system. Resonances of GC11, T54-m1A58, GU4, and A psi 31 were confirmed, assigned, or reassigned. psi 39 was found to be in anti conformation, not syn as previously stated. Almost all the uracil NH group resonances could be separated, but most of the GC resonances are too close even in two dimensions to be separately resolved with the observed 20-Hz 15N line width.

Guanosine↗
PubMed1983

Proton exchange rates in transfer RNA as a function of spermidine and magnesium.

Solvent exchange rates of selected protons were measured by NMR saturation recovery for E. coli tRNAVal, E. colifMet and yeast tRNAPhe, at temperatures from 20 to 40 degrees C, in the presence of 0.12M Na+ and various levels of added spermidine. tRNAVal was also studied with added Mg++. The exchange rates in zero spermidine and Mg++ indicate early melting of the U8 A14 interaction, in accord with thermodynamic melting studies. Exchange rates for secondary protons suggest early melting of the T stem in tRNAfMet and the acceptor stem in tRNAPhe, in contradiction with melting transition assignments from thermodynamic work. Addition of 10 spermidines per tRNA stabilizes the secondary and tertiary interactions more effectively than added Na+, but less so than Mg++. Added spermidine has the curious effect of increasing the exchange rate of the psi 55 N1 proton, while protecting the psi 55 N3 proton from exchange in all three tRNA's. Added Mg++ has the same effect on tRNAVal.

Escherichia coli↗
PubMed1983

Assignment of imino proton spectra of yeast phenylalanine transfer ribonucleic acid.

Yeast tRNAPhe has been studied by using proton NMR and nuclear Overhauser effect (NOE) with deuterium substitution. Direct NOE evidence is presented for assignment of imino resonances of 23 of 27 base pairs in this tRNA. Other indirect evidence is presented for tentative assignment of four other base pairs. Almost total assignment also has been made of the important noninternally bonded imino protons and tertiary interactions (however, G18-psi 55 remains unassigned). The most surprising result has been identification of GC11 at -13.68 ppm; this is the first time a GC base pair has been identified so far downfield. This peak (GC11) is also identified as the resonance of the unique imino proton that exchanges in a time of more than 1 day, as previously described. These identifications of imino proton resonances made it possible to reinterpret the proton solvent exchange rate data previously published on this tRNA and understand them better. The assignments of resonances should pave the way for more detailed solution study of this tRNA and its interaction with biologically relevant molecules.

Anticodon↗
PubMed1982

Nuclear Overhauser effect study of yeast tRNAVal 1: evidence for uridine-pseudouridine base pairing.

The proton NMR spectrum of yeast tRNAVal 1 has been studied using nuclear Overhauser effect (NOE), including comparison of NOE patterns between purine C8 deuterated and nondeuterated samples. Studies of the downfield region enable us to reliably assign many resonances in the acceptor and D stems. Prominent among these reliable assignments is that of the unusual base pair U psi, which is made here for the first time. Other identifications include GU2, U8-A14, the three AU base pairs of the acceptor stem, and N1 and N3 protons of psi 55.

Base Composition↗
PubMed1982

Procedure for C2 deuteration of nucleic acids and determination of A psi 31 pseudouridine conformation by nuclear Overhauser effect in yeast tRNAPhe.

Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA. Deuterated tri-ethyl orthoformate was condensed with 5(4)-amino imidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine. C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated yeast tRNAPhe was isolated. This C2 deuterated tRNAPhe was used to identify A psi 31 and U8-A14. A psi 31 was found to be bonded through N1H. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.

Base Sequence↗
PubMed1982

Nuclear overhauser effect study of yeast aspartate transfer ribonucleic acid.

Nuclear Overhauser effect studies are described for yeast tRNAAsp in 0.1 M NaCl, pH 7.0. A primary aim is to develop a general method for attacking the problem of assignment in transfer ribonucleic acids (tRNAs). Previously, we have demonstrated the utility of the nuclear Overhauser effect (NOE) between protons on adjacent base pairs combined with C8 deuterium substitution, by assigning the imino protons of the dihydrouridine stem and the two reverse-Hoogsteen base pairs T54-A58 and U8-A14. Here, we extend that approach to other parts of the molecule. We also describe several NOE-connected patterns for, e:g., m5CG and psi 55 N3H imino protons which may be of general utility. For the first time, a purine-15-pyrimidine-48 base pair (in this case A15-U48) has been assigned. A total of 13 of 25 base pairs from all parts of the molecule and several noninternally bonded imino protons have now been assigned unambiguously. This is a general method for assigning resonances in tRNA and perhaps in all double-stranded nucleic acids. This, and the distance information inherent in NOE measurements, should make NMR more generally applicable to nucleic acids.

Base Sequence↗
PubMed1981

Nuclear Overhauser effect study and assignment of D stem and reverse-Hoogsteen base pair proton resonances in yeast tRNAAsp.

Nuclear Overhauser effects (NOEs) in yeast tRNAAsp were found for all four GU and G psi base pairs. NOEs of both reverse-Hoogsteen pairs were identified by comparison with a purine C8 deuterated sample. Several NOEs involving these resonances were also found which are clearly between single protons on adjacent base pairs. These interbase NOEs, combined with the assumption of reasonable similarity between the structure of yeast tRNAAsp and that of yeast tRNAPhe, lead to unambiguous assignment of many resonances including all the ring NH and C2 protons in the D stem. The stability of the stem at 28 degrees C, as recently deduced by Moras et al (Nature 288 669-674), from x-ray diffraction is confirmed. Assignments of the ring NH resonances of T54-A58 and of a G psi pair are made for the first time.

Base Composition↗
PubMed1981

Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance.

Nuclear magnetic resonance (NMR) measurements of proton exchange were performed on yeast tRNAPhe, and in much less detail on Escherichia coli tRNAfMet, over a range of Mg2+ concentrations and temperatures, at neutral pH and 0.1 M NaCl. The resonances studied were those of ring nitrogen protons, resonating between 10 and 15 ppm downfield from sodium 3-(trimethylsilyl)-1-propanesulfonate, which partake in hydrogen bonding between bases of secondary and tertiary pairs. Methods include saturation--recovery, line width, and real-time observation after a change to deuterated solvent. The relevant theory is briefly reviewed. We believe that most of the higher temperature rates reflect major unfolding of the molecule. For E. coli tRNAfMet, the temperature dependence of the rate for the U8--A14 resonance maps well onto previous optical T-jump studies for a transition assigned to tertiary melting. For yeast tRNAPhe, exchange rates of several resolved protons could be studied from 30 to 45 degrees C in zero Mg2+ concentration and had activation energies on the order of 40 kcal/mol. Initially, the tertiary structure melts, followed shortly by the acceptor stem. At high Mg2+ concentration, relatively few exchange rates are measurable below the general cooperative melt at about 60 degrees C; these are attributed to tertiary changes. Real-time observations suggest a change in the exchange mechanism at room temperature with a lower activation energy. The results are compared with those obtained by other methods directed toward assaying ribonucleic acid dynamics.

Escherichia coli↗