1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Thériault, Yves; Pochapsky, Thomas C.; Dalvit, Claudio; Chiu, Mark L.; Sligar, Stephen G.; Wright, Peter E.

doi:10.1007/bf00156616

Cited by 45 publications

(30 citation statements)

References 50 publications

(38 reference statements)

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“…For myoglobin, the structure is known (18) and assignments for the diamagnetic carboxy form of the protein at pH 5.6 and 35°C are available (12). We can, therefore, approach assignment in a slightly different way.…”

Section: Resultsmentioning

confidence: 99%

“…Several groups have now shown that high-resolution NMR experiments, including multidimensional experiments normally used for resonance assignment, are applicable to certain paramagnetic proteins (10,11). In the case of myoglobin, the recent publication of a complete set of assignments for 'H and 15N resonances in the diamagnetic carboxy form also may aid assignment by allowing the correlation of chemical shifts and NOE connectivity patterns in the diamagnetic versus paramagnetic states (12).…”

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confidence: 99%

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Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

Tolman

Flanagan

Kennedy

et al. 1995

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

815

701

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The measurement ofdipolar contributions to the splitting of 15N resonances of 1H-15N amide pairs in multidimensional high-field NMR spectra of field-oriented cyanometmyoglobin is reported. The splittings appear as small field-dependent perturbations of normal scalar couplings. Assignment of more than 90 resonances to specific sequential sites in the protein allows correlation of the dipolar contributions with predictions based on the known susceptibility and known structure of the protein. Implications as an additional source of information for protein structure determination in solution are discussed.Within the past 15 years, NMR has emerged as a powerful approach to the study of protein structure in solution (1, 2). The approach relies on distance constraints extracted from nuclear Overhauser effects (NOEs) and typically proceeds in three stages: assignment of backbone resonances to specific sequential sites, identification of secondary structure elements, and determination of a tertiary fold. The latter stage is particularly demanding because it requires that a large number of long-range distance constraints be extracted from NOE data and assigned to specific proton pairs. Because NOEs drop off with the inverse 6th power of the internuclear distance, the pairs tend to arise from direct side-chain-side-chain contacts, contacts involving protons which are among the most difficult to assign. Opportunities to supplement long-range distance constraints with other types of structural data would, therefore, be welcome. We demonstrate here that NMR spectra of certain proteins, taken at very high field, may contain data that can usefully complement NOEs in determining a tertiary fold. The data come from residual dipolar contributions to the scalar couplings normally seen in high-resolution spectra. These appear when the protein has a slightly preferred orientation in a magnetic field. The contributions are angle dependent and can yield constraints for the orientation of one structural element relative to another structural element. In our case, we use a 17.5-kDa protein, cyanometmyoglobin, which has a very highly anisotropic paramagnetic susceptibility to achieve preferred orientation. Myoglobin crystals have been previously shown to orient in a magnetic field because of their anisotropy (3). Bothner-By and co-workers (4) also showed years ago that orientational effects on NMR spectra of single molecules in solution could be observed if fields and resolution were high enough. That these effects can be observed in an isolated protein molecule, and that the effects can provide useful structural constraints, has awaited higher fields (17.5 T) and multidimensional NMR experiments for the detection and assignment of 15N resonances in an isotopically labeled protein.Theory. The dipolar interaction between two spin 1/2 nuclei in the high-field limit is given by the formulawhere the -y values are the gyromagnetic ratios for the nuclei, h is Plank's constant, r is the distance between the nuclei, 0 is the angle between...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

Tolman

Flanagan

Kennedy

et al. 1995

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

815

701

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“…2B!. The MbCO chemical shifts~Kao & Lecomte, 1993;Osapay et al, 1994;Thèriault et al, 1994! were corrected for the heme ring current contribution as described elsewhere~Kao & Lecomte, 1993!.…”

Section: Resultsmentioning

confidence: 99%

Conformational properties of native sperm whale apomyoglobin in solution

et al. 1999

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Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear~1 3 C and 15 N! NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects NOEs! involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid~ANS! were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3J aH-NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X-PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H-helix. The EF-F-FG segment remains uncharacterized.

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“…2, traces A and C), disappear from the indicated spectral region of the corresponding apoproteins. If, as judged from their shifts, they arise from GluC3 amide NH proton in the holoproteins [28], these results strongly suggest that a significant structural alteration in the C helix is induced upon heme removal. GluC3 amide NH proton signal of whale Mb (at 35°C and pH .5.6), observed at 11.03 ppm [28], shifted to 10.51 ppm in the spectrum of the apoprotein (at 25°C and pH 5.7) [29].…”

Section: Resultsmentioning

confidence: 92%

“…Peaks a, and b, have been assigned to HisEF5 NeH and HisB5 N6H protons, respectively, and peak c, was tentatively assigned to HisFG3 NeH proton 1271. Based on the observed shift, peak d, could be attributed to GluC3 amide NH proton [28]. In the spectrum of whale apoMb (Fig.…”

Section: Resultsmentioning

confidence: 93%

¹H‐NMR Study of Inter‐Segmental Hydrogen Bonds in Sperm Whale and Horse Apomyoglobins

Yamamoto

1997

European Journal of Biochemistry

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NMR signals for HisBS N6H and HisEFS NeH protons of sperm whale and horse apomyoglobins were assigned and compared with the corresponding signals of the holoproteins in terms of pHand temperature dependence behaviors of their shifts and line widths in order to gain insight into structural difference between the apoproteins and the holoproteins. Since these protons are involved in internal hydrogen bonds at the interfaces between the B helix and the GH corner and between the EF corner and the H helix, local structures of the interfaces in these proteins have been inferred from the analyses of these signals. A large difference in the line width of HisEF5 NeH proton signal between the apoproteins and the holoproteins strongly suggested that a sizable structural alteration is induced in the EF-H interface by the removal of heme. However, the results for HisBS N6H proton resonance indicated the absence of a significant structural alteration in the B-GH interface by heme extraction. These results are consistent with the data obtained from mutation [Hughson, F. M.

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1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Cited by 45 publications

References 50 publications

Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

Conformational properties of native sperm whale apomyoglobin in solution

¹H‐NMR Study of Inter‐Segmental Hydrogen Bonds in Sperm Whale and Horse Apomyoglobins

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1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Cited by 45 publications

References 50 publications

Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

Conformational properties of native sperm whale apomyoglobin in solution

1H‐NMR Study of Inter‐Segmental Hydrogen Bonds in Sperm Whale and Horse Apomyoglobins

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Product

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¹H‐NMR Study of Inter‐Segmental Hydrogen Bonds in Sperm Whale and Horse Apomyoglobins