Determination of the molecular conformation of melanostatin using carbon-13, nitrogen-15 REDOR NMR spectroscopy

Garbow, Joel R.; McWherter, Charles A.

doi:10.1021/ja00054a034

Cited by 59 publications

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“…Both corrections are done analytically assuming standard peptide geometry and bond lengths. 7 After corrections, the experimentally determined ratio AS/ S 0 is 0.159. Reference to Further REDOR experiments with different labeling schemes are planned to probe the conformation of model compounds of silk I.…”

Section: Methodsmentioning

confidence: 98%

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Structure of Bombyx mori Silk Fibroin Studied by REDOR NMR Spectroscopy

Asakura

Aoki

Demura

et al. 1994

Polym J

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KEY WORDSBombyx mori Silk Fibroin / Rotational-Echo / DoubleResonance / (REDOR) NMR / Anti-Parallel /3-Sheet / 13 C-15 N dipolar Interaction / It has been confirmed that the solid-state conformation of silk fibroin from Bombyx mori is strongly dependent on sample preparation. 1 It is well known that the silk II form (antiparallel /)-sheet), obtained by drying at high temperature or under mechanical stress, is the most stable form. The metastable silk I form is obtained by drying from aqueous solution at room temperature. 13 C chemical shifts are sensitive to molecular conformation and have been used to show that silk I and silk II are structurally different. 1 Whereas the structure of silk II has been known for some time ( for example, see ref 2), the structure of silk I has not been determined due, primarily, to the difficulty in obtaining high quality oriented samples. However, several models have been proposed for the silk I structure. These include the "crankshaft" model by Lotz and Keith 2 and three models by Asakura and Yamaguchi 3 based on 13 C CP/MAS NMR, X-ray diffraction data and conformational energy considerations.We are initiating efforts to obtain the backbone conformation of silk I using the t To whom requests for reprints should be addressed. rotational-echo, double-resonance (REDOR)NMR technique to measure internuclear distances between specific 13 C-15 N spin pairs. 4 -6 These measured distances will be used to determine the dihedral angles describing the backbone conformation. 7 A particular advantage of our strategy is that oriented samples are not required and simple powder samples can be used. As a demonstration of the technique, we present initial REDOR results on a model compound with the well-characterized silk II conformation. THEORETICAL BACKGROUNDRotational-echo, double-resonance NMR is a high-resolution, solid-state MAS NMR technique designed to measure internuclear distances between heteronuclear spin pairs. 4 -6 REDOR is ideally suited for measuring specific internuclear distances between selectively labeled spin sites. In particular, selective labeling of proteins and peptides with 13 C and 15 N isotopes and measuring the internuclear separation between the labeled spin pair is 1405

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Section: Methodsmentioning

confidence: 98%

“…The measured 13 C-15 N distance will place limits on the possible values of the <p, t/J dihedral angles of the Ala 5 residue. 7 The REDOR experiments were performed at a 13 C frequency of 38.07 MHz at Florida 'State University on a home-built spectrometer. The 230 mg sample was centered in a zirconia rotor with Kel-F spacers.…”

Section: Methodsmentioning

confidence: 99%

Structure of Bombyx mori Silk Fibroin Studied by REDOR NMR Spectroscopy

Asakura

Aoki

Demura

et al. 1994

Polym J

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“…This sequence is applied in synchrony with MAS, and contains excitation, evolution, mixing, and detection periods. In the excitation period, transverse 13 C-spin polarization is first created by cross-polarization 16 XY-16 phases, 18 is applied for a total time N 1 R . A Gaussianshaped, frequency-selective 13 C pulse with duration M R is then applied to carbonyl spins, and a hard 15 N pulse is applied at the midpoint of the frequency-selective 13 C pulse.…”

Section: Theory and Pulse Sequencementioning

confidence: 99%

“…At the end of this excitation period, the spin-density operator for an 15 N-13 C pair is ideally proportional to I x cos 2d 1 R š 2I y S y sin 2d 1 R , where I˛and S˛are angular momentum component operators for 13 19 ), while simultaneously refocusing inhomogeneous broadening and scalar couplings of the labeled 13 CO sites, as in the frequency-selective REDOR technique of Jaroniec et al 20 The evolution period contains similar REDOR pulse trains and selective 13 C inversion, but with the durations of the REDOR trains set to N 2 R and with additional N 3 R delays at the beginning and end of the evolution period. Because the DSQ coherence for the 13 13 C pulse in the evolution period, and homonuclear dipole-dipole couplings are removed by MAS. Thus, dephasing of the DSQ coherence depends on the angle between directions R i and R i+1 shown in Fig.…”

Section: Theory and Pulse Sequencementioning

confidence: 99%

“…A number of solid-state NMR techniques that directly constrain i and i in unoriented samples under magic-angle spinning (MAS) have been demonstrated. Some of these are intended for applications to samples with 13 C or 15 N labels at specific sites. 1 -8 Others are applicable to samples with uniform 15 N and 13 C labeling of at least two sequential amino acid residues, 9 or samples with isolated uniformly labeled residues.…”

Section: Introductionmentioning

confidence: 99%

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Conformational constraints in solid-state NMR of uniformly labeled polypeptides from double single-quantum-filtered rotational echo double resonance

Oyler

Tycko

2007

Magn. Reson. Chem.

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A solid-state nuclear magnetic resonance (NMR) technique is described for obtaining constraints on the backbone conformation of a protein or peptide that is prepared with uniform (15)N,(13)C labeling of consecutive pairs of amino acids or of longer segments. The technique, called double single-quantum-filtered rotational echo double resonance (DSQ-REDOR), uses frequency-selective REDOR to prepare DSQ coherences involving directly bonded backbone (13)CO and (15)NH sites, to dephase these coherences under longer-range (15)NH-(13)CO dipole-dipole couplings in a conformationally dependent manner, and to convert the remaining DSQ coherences to detectable transverse (13)C-spin polarization. The efficacy of DSQ-REDOR is demonstrated in experiments on two isotopically labeled samples, the helical peptide MB(i + 4)EK and the amyloid-forming peptide Abeta(11-25).

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Accuracy Limitations on Internuclear Distances Measured by REDOR

Naito

Saitô

2007

Encyclopedia of Magnetic Resonance

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Determination of the molecular conformation of melanostatin using carbon-13, nitrogen-15 REDOR NMR spectroscopy

Cited by 59 publications

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Structure of Bombyx mori Silk Fibroin Studied by REDOR NMR Spectroscopy

Structure of Bombyx mori Silk Fibroin Studied by REDOR NMR Spectroscopy

Conformational constraints in solid-state NMR of uniformly labeled polypeptides from double single-quantum-filtered rotational echo double resonance

Accuracy Limitations on Internuclear Distances Measured by REDOR

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