Pulsed NMR methods for the observation and assignment of exchangeable hydrogens: Application to bacitracin

Campbell, Iain D.; Dobson, Christopher M.; Jeminet, Georges; Williams, R. J. P.

doi:10.1016/0014-5793(74)80645-9

Cited by 101 publications

(28 citation statements)

References 14 publications

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“…Both spectrometers were operated in the Fourier transform mode, using a Nicolet 1085 computer. The observation of the amino protons of 5'-AMP, 5'-GMP and 5'-CMP, carried out in H20, was made in the Fourier transform mode, using the gated decoupling technique to suppress the large solvent peak [7]. The internal standard was sodium 3-trimethyl~ily1 [ All the nucleotides (from Sigma Chemical Company) and the disodium salt of EDTA (from B.D.H.…”

Section: Methodsmentioning

confidence: 99%

Nuclear-Magnetic-Resonance Studies of 5'-Ribonucleotide and 5'-Deoxyribonucleotide Conformations in Solution Using the Lanthanide Probe Method

et al. 1978

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icleo tide WILLIAMSThe conformations of the metal-bound 5 '-ribonucleotides and 5 '-deoxyribonucleotides in aqueous solution at different pH values have been studied using the lanthanide probe method. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from coupling constants, nuclear Overhauser effects and spin-lattice relaxation times, obtained for the metal-fixed systems. The equilibrium between the two basic conformational combinations for the 5 '-nucleotides, anti-(N G? S ) -gg-g'g' and syn-(NG?S)-gt-g'g' depends on the nature of the furanose ring, the base and also on the state of base protonation and phosphate ionization. The effect of base protonation is particularly strong for the guanine nucleotides.The conformations of free 5 '-ribonucleotides and deoxyribonucleotides in solution have been extensively studied by nuclear magnetic resonance (NMR) spectroscopy, due to the importance of these molecules as the simplest constituents of naturally occurring nucleic acids. Most of these studies have been based on the interpretation of experimental NMR parameters for the isolated nucleotides, such as chemical shifts, vicinal spin-spin coupling constants, nuclear Overhauser enhancements and nuclear relaxation rates [I, 11 -13,281 (and references in [I]). A different general method for the determination of the conformation of metal-bound flexible molecules in solution, using the lanthanide cations (Ln3 ') as shift and relaxation NMR probes has been described and applied to some nucleotide monophosphatates [2-41.In this paper the lanthanide probe method is applied to the comparative study of the conformations in aqueous solution of the eight metal-bound 5'-mOnOnucleotides which occur in ribonucleic and deoxyribonucleic acids : the nucleotides of adenosine (5'-AMP), guanosine (5'-GMP), cytidine (5'-CMP), uridine (5 '-UMP), 2'-deoxyadenosine (5 '-dAMP), 2'-deoxyguanosine (5'-dGMP), 2'-deoxycytidine (5'-dCMP) and thymidine (5'-dTMP).The conformational conclusions obtained from the lanthanide data are compared with independent NMR Ahbrevintion. NMR, nuclear magnetic resonance work, especially coupling constants and nuclear Overhauser enhancement data, as there must be at least a qualitative agreement for the metal-bound systems between the conclusions from the different methods [5,6]. From this comparison, an assessment of the different NMR conformational methods in solution is attempted. Finally the results in aqueous solution are compared with the available information on the crystal structure of metal-bound 5 '-mononucleotides from X-ray diffraction data and with the data on the metal-free systems. MATERIALS AND METHODS'H NMR spectra were obtained at 90 MHz using a Bruker HFX 90 spectrometer and at 270 MHz using an Oxford Instruments superconducting magnet and a modified Bruker HFX 90 console. Both spectrometers were operated in the Fourier transform mode, using a Nicolet 1085 computer. The observation o...

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Section: Methodsmentioning

confidence: 99%

Nuclear-Magnetic-Resonance Studies of 5'-Ribonucleotide and 5'-Deoxyribonucleotide Conformations in Solution Using the Lanthanide Probe Method

et al. 1978

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“…The solvent peak was saturated with a second frequency and a third frequency was used in the transfer of saturation experiments [10]. The second and third frequencies were applied at all times except during data acquisition.…”

Section: Methodsmentioning

confidence: 99%

Cross correlation of titrating histidines in oxy‐ and deoxyhaemoglobin; an NMR study

Brown

Campbell

1976

FEBS Letters

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Haemoglobin is the best known example of a protein exhibiting co-operative binding properties and continues to excite active interest [1][2][3]. One feature of the co-operative behaviour is the release of protons on binding oxygen, a phenomenon known as the Bohr effect. Perutz in his classic X-ray work [4] identified three groups which might be involved in the Bohr effect and Kilmartin et al. [5] in an n.m.r. study of CO and deoxy forms of haemoglobin confirmed that one of these groups was His/3146. Val al has also been identified, by chemical modification studies [6], as another group involved but there is still uncertainty about the role of other groups, although these are expected to be histidines. N.m.r. seems to be the technique most likely to provide the detailed information about ionization states necessary to resolve this problem, but at present it is limited by difficulties in resolving and assigning the titrating histidine resonances.The key factor in an n.m.r, study of the involvement of histidines in the Bohr effect lies in the correlation of peaks in oxy-and deoxyhaemoglobin. In this communication we illustrate a powerful method for correlating peaks whose movements in an oxydeoxy titration are discontinuous and therefore impossible to follow by stepwise titration procedures. The method, which has been used previously to correlate resonances of reduced and oxidised cytochrome c [7] requires that the rate of exchange between liganded and unliganded forms be faster than the nuclear spin lattice relaxation rate (l/T1). This requirement is shown to be met for at least one histidine resonance in haemoglobin when oxygen is the ligand.

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“…It remains to be demonstrated whether the slowest exchanging protons in cytochrome c are associated with d structure in the protein. The NMR spectra of reduced cytochrome c recorded under conditions of solvent suppression (26) and resolution enhancement (25) permit the observation of additional resonances in this spectral region (Fig. 1).…”

Section: Spectramentioning

confidence: 99%

Nuclear magnetic resonance studies of slowly exchanging peptide protons in cytochrome c in aqueous solution.

Patel¹,

Canuel²

1976

Proc. Natl. Acad. Sci. U.S.A.

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The slowly exchanging protons in oxidized and reduced horse heart cytochrome c (D20, uncorrected pH meter reading 6.5, room temperature) have been monitored by recording the 270 and 360 MHz proton nuclear magnetic resonance spectra of the reduced protein between 5 and 11 parts per million downfield from 2,Aimethyl-2-silapentane-5-sulfonate.There are about 12 well-resolved exchangeable resonances between 7.9 and 10.1 parts per million and the experimental chemical shift data suggest that they originate from the same residues in both oxidation states. These resonances correspond to 19-22 protons in the reduced protein and a lesser number due to partial exchange in the oxidized protein. The base catalysis of the exchange in both oxidation states was used to demonstrate the sequential exchange of these well-resolved resonances in D20. Alternatively, the temperature dependence of the exchange at a fixed pH was used to differentiate and monitor separately the slower and the slowest exchanging resonances as a function of time. The different rates of exchange among the slowly exchanging peptide protons provide evidence against a resistant cluster of protons that exchange together. Cytochrome c has been studied extensively by chemical and physical techniques with the eventual goal of understanding its role in coupled electron transport (1, 2). X-ray crystallographic studies of the oxidized and reduced cytochrome c have added considerably to our knowledge of the polypeptide conformation and heme group contacts in the crystal (3, 4). Physiological mechanisms have been proposed for the oxidoreduction of cytochrome c based on the available x-ray data on the cytochrome class of proteins (5, 6).Nuclear magnetic resonance (NMR) spectroscopic studies have elucidated many aspects of the cytochrome c -structure in solution (7-12, and Exchange Studies on the Oxidized Protein. Oxidized cytochrome c was dissolved in H20, the pH was adjusted to 6.5, and the solution was lyophilized. The lyophilized material was dissolved in D20 to an 8 mM protein concentration and the pH (uncorrected pH meter reading) was raised to the desired value with NaOD. The samples were heated in a water bath at a given temperature and the aliquots were removed as a function of time. The oxidized samples were reduced with dithionite and the NMR spectra were recorded on the reduced cytochrome c in D20 solution.Exchange Studies on the Reduced Protein. Oxidized cytochrome c was dissolved in H20 solution, the pH was adjusted to 6.5, and the protein was reduced with dithionite. The reduced protein in H20 at pH 6.5 was lyophilized, then dissolved in D20 solution to an 8 mM protein concentration, and the uncorrected pH meter reading was changed to the desired value with NaOD. The samples were heated in the water bath at the desired temperature and aliquots of the reduced protein were removed as a function of time to record the NMR spectra. Proton NMR spectra

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Pulsed NMR methods for the observation and assignment of exchangeable hydrogens: Application to bacitracin

Cited by 101 publications

References 14 publications

Nuclear-Magnetic-Resonance Studies of 5'-Ribonucleotide and 5'-Deoxyribonucleotide Conformations in Solution Using the Lanthanide Probe Method

Nuclear-Magnetic-Resonance Studies of 5'-Ribonucleotide and 5'-Deoxyribonucleotide Conformations in Solution Using the Lanthanide Probe Method

Cross correlation of titrating histidines in oxy‐ and deoxyhaemoglobin; an NMR study

Nuclear magnetic resonance studies of slowly exchanging peptide protons in cytochrome c in aqueous solution.

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