Conformation and segmental motion of native and denatured ribonuclease A in solution. Application of natural-abundance carbon-13 partially relaxed Fourier transform nuclear magnetic resonance

Allerhand, Adam; Doddrell, David M.; Glushko, Victor; Cochran, David W.; Wenkert, Ernest; Lawson, Peter J.; Gurd, Frank R. N.

doi:10.1021/ja00731a053

Cited by 190 publications

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“…Eq. 2 is valid when Tefl is much smaller than the inverse of the proton and carbon resonance frequencies, a condition which holds for the solutions studied here, as can be shown by means of linewidth measurements (9). In a rigid fused-ring system, Teff is about the same for all ring-carbons.…”

Section: Resultssupporting

confidence: 57%

“…The former is often complicated by uncertainties in chemical shift changes with structure; the latter is difficult to use when there are many overlapping lines (5). We show below that 13C partially-relaxed Fourier transform (PRFT) spectra (8,9) provide additional help in making assignments. Intensities in PRFT spectra are given (10) by A = Ao [1 -2 exp(-rT1)],…”

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confidence: 97%

“…where A and Ao are the observed and equilibrium intensities, described previously (7,9). When using the 180'-T-90' pulse sequence (10) needed to obtain PRFT spectra (8,9), we chose the recycle time between sequences to be at least three times the longest T1 value of interest.…”

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Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B ₁₂ , Coenzyme B ₁₂ , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy

Doddrell

Allerhand

1971

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

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High-resolution Fourier transform NMR at 15.08 MHz was used to observe the proton-decoupled natural-abundance 13C spectra of aqueous solutions of cobinamide dicyanide (0.067 M), cyanocobalamin (0.024 M), dicyanocobalamin (0.14 M), and coenzyme B12 (0.038 M). Assignments were made with the aid of chemical shift comparisons, off-resonance single-frequency proton decoupling, partially-relaxed Fourier transform spectra, and splittings arising from 13C-31P coupling.As expected, the 13C spectra of the corrinoids were appreciably more informative than the corresponding proton spectra. Nearly all the lines in the 13C spectra of the corrinoids were well-resolved single-carbon resonances, in spite of the structural complexity.Partially relaxed 13C Fourier transform NMR spectra, which yield spin-lattice relaxation times of each resolved resonance, were found to be a very useful addition to the arsenal of NMR techniques.Proton nuclear magnetic resonance (NMR) has been useful for investigating the solution properties of vitamin B12 (cyanocobalamin, Fig. 1A) and other corrinoids (1-4). However, even at 220 MHz, proton NMR spectra of corrinoids contain many overlapping lines, as a result of a small range of chemical shifts and complex spin-spin splittings (4). For the study of complex molecules, proton-decoupled spectra of 13C nuclei in natural abundance are, in general, much more resolved and simpler to analyze than the corresponding proton spectra (5). The enhanced sensitivity of the Fourier transform technique (6) relative to continuous-wave N1\IR has expanded the range of accessible 13C spectra (7). We will show that by using the Fourier transform method it is easy to obtain high signal-to-noise ratios on single-carbon resonances in the proton-decoupled natural-abundance 13C spectra of corrinoids, even when the concentration is limited to 0.02 M.There are only two well-known generally applicable aids to the assignment of 13C resonances in complex molecules: comparisons within a series of compounds with similar structures, and the use of splitting patterns arising from incomplete proton-decoupling (5). The former is often complicated by uncertainties in chemical shift changes with structure; the latter is difficult to use when there are many overlapping lines (5). We show below that 13C partially-relaxed Fourier transform (PRFT) spectra (8, 9) provide additional help in making assignments. Intensities in PRFT spectra are given (10) by A = Ao [1 -2 exp(-rT1)],

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Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B ₁₂ , Coenzyme B ₁₂ , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy

Doddrell

Allerhand

1971

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

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“…The sharpening of resonance lines for denatured as compared with native hen egg-white lysozyme reflect the influence of the tertiary structure and the disulphide bridges of the native protein ( Fig.5 and 6). The degree to which the loss of tertiary structure versus the reduction of the disulphide bridges contributes to the narrower resonances for the side chains is uncertain, although the results of Allerhand et al [47] for the acid denaturation of RNAase A (disulphide bridges intact) indicate that the a-and /?-carbon resonances are significantly broadened by tertiary effects.…”

Section: I3c-nmr Studies Of Amino Acids Peptides and Proteinsmentioning

confidence: 99%

Carbon‐13 Nuclear‐Magnetic‐Resonance Studies on Selected Amino Acids, Peptides, and Proteins

Freedman

Lyerla

Chaiken

et al. 1973

European Journal of Biochemistry

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Natural abundance high-resolution carbon-13 Fourier transform nuclear magnetic resonance (W NMR) studies a t 25 MHz have been carried out on selected amino acids, linear peptides and proteins, The pH dependence of the 13C resonances has been studied in detail in several cases. For L-histidine, the 18C chemical shifts of all six carbon atoms are sensitive to the ionization state of the titrating carboxyl, imidazole and amino groups. The data for each carbon resonance have been computer-fitted to a sum of three theoretical curves based on a simple proton-association equilibrium for each transition. The extent of chemical shift change upon ionization of a nearby group has been interpreted in terms of two competing effects, inductive (through-bond) and electric field (through-space) effects.NMR spectra of the amino-terminal 1-13, 1-15, and 1-20 peptides of bovine pancreatic ribonuclease A have been analyzed. Detailed resonance assignments have been made based on comparisons with the results for the free amino acids, shift effects for amino acids in small peptides, internal peptide bond effects and differences in amino acid composition. There is considerable correspondence between the observed 13C chemical shifts of these peptides and those predicted from the shifts of the component amino acids.Proteins of varying molecular weight have been studied to evaluate the potential of 1% NMR investigations of structure and conformation. A comparison has been made between the 18C NMR spectra of hen egg-white lysozyme in its native and reduced states. The spectrum of the fully reduced species appears to be well represented by the sum of the spectra of the individual amino acids, both in chemical shift and line width, whereas that for the native form exhibits consistently broader resonances. Similar results were obtained for ribonuclease A. At the current level of resolution, it is impossible to discern whether this line broadening derives from shorter relaxation hime effects due to restricted motion, chemical shift non-equivalence between the same chemical groups, or both. However, it does appear that there are separately resolved peaks corresponding to CB-carbons for different threonyl residues, as well as for the C-4 ring carbons of the several tyrosyl residues in native hen egg-white lysozyme. Spectra of carbonic anhydrases (Mr 4 30000), which lack disulphide bonds, showed somewhat poorer resolution than the smaller proteins. TheIn recent years, there has been considerable interest in the potential use of nuclear magnetic resonance (NMR), particularly proton magnetic resonance (lH NMR), for characterizing protein conformation and interactions [l -31. In principle, lH NMR spectra of proteins contain a wealth of

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“…It was predicted that differential saturation of specific'carbons would not be a problem using a 900 pulse with a total pulse delay and acquisition time of 0.74 seconds from the molecular weight range of the fulvic acid samples and 13C nmr studies of other macromolecules with similar molecular weights .specifically cholesterol (Allerhand Doddrell, and Komoroski, 1971) and low molecular weight polypeptides (Allerhand, and Komoroski, 1973;Deslauriers, Smith,and Walter, 1974;Keim, Vigna, Marshall, and Gurd, 1973; and proteins (Allerhand, Doddrell, Glushko, Cochran, Wenkert, Lawson, and Gurd, 1971;Glushko, Lawson, and Gurd, 1972;Allerhand, Childers, and Oldfield, 1973;Hunkapiller, Smallcombe, Whitaker, and Richards, 1973)]. …”

Section: Discussionmentioning

confidence: 99%

The characterization of humic substances in seawater

Stuermer¹

1975

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Conformation and segmental motion of native and denatured ribonuclease A in solution. Application of natural-abundance carbon-13 partially relaxed Fourier transform nuclear magnetic resonance

Cited by 190 publications

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Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B ₁₂ , Coenzyme B ₁₂ , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy

Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B ₁₂ , Coenzyme B ₁₂ , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy

Carbon‐13 Nuclear‐Magnetic‐Resonance Studies on Selected Amino Acids, Peptides, and Proteins

The characterization of humic substances in seawater

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Conformation and segmental motion of native and denatured ribonuclease A in solution. Application of natural-abundance carbon-13 partially relaxed Fourier transform nuclear magnetic resonance

Cited by 190 publications

References 0 publications

Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B 12 , Coenzyme B 12 , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy

Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B 12 , Coenzyme B 12 , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy

Carbon‐13 Nuclear‐Magnetic‐Resonance Studies on Selected Amino Acids, Peptides, and Proteins

The characterization of humic substances in seawater

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Product

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Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B ₁₂ , Coenzyme B ₁₂ , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy

Assignments in the Carbon-13 Nuclear Magnetic Resonance Spectra of Vitamin B ₁₂ , Coenzyme B ₁₂ , and Other Corrinoids: Application of Partially-Relaxed Fourier Transform Spectroscopy