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. 1994 Feb;3(2):267–281. doi: 10.1002/pro.5560030211

The native state of apomyoglobin described by proton NMR spectroscopy: interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS.

M J Cocco 1, J T Lecomte 1
PMCID: PMC2142796  PMID: 8003963

Abstract

Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His-64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His-82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481-6491). The packing of the side chains and the low pKa of His-64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter-amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.

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Selected References

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  1. Barrick D., Baldwin R. L. Three-state analysis of sperm whale apomyoglobin folding. Biochemistry. 1993 Apr 13;32(14):3790–3796. doi: 10.1021/bi00065a035. [DOI] [PubMed] [Google Scholar]
  2. Breslow E., Koehler R., Girotti A. W. Properties of protoporphyrin-apomyoglobin complexes and related compounds. J Biol Chem. 1967 Sep 25;242(18):4149–4156. [PubMed] [Google Scholar]
  3. Brooks C. L., 3rd Characterization of "native" apomyoglobin by molecular dynamics simulation. J Mol Biol. 1992 Sep 20;227(2):375–380. doi: 10.1016/0022-2836(92)90893-o. [DOI] [PubMed] [Google Scholar]
  4. Cocco M. J., Kao Y. H., Phillips A. T., Lecomte J. T. Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy. Biochemistry. 1992 Jul 21;31(28):6481–6491. doi: 10.1021/bi00143a018. [DOI] [PubMed] [Google Scholar]
  5. Dalvit C., Wright P. E. Assignment of resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of sperm whale myoglobin by phase-sensitive two-dimensional techniques. J Mol Biol. 1987 Mar 20;194(2):313–327. doi: 10.1016/0022-2836(87)90378-0. [DOI] [PubMed] [Google Scholar]
  6. Dill K. A. Dominant forces in protein folding. Biochemistry. 1990 Aug 7;29(31):7133–7155. doi: 10.1021/bi00483a001. [DOI] [PubMed] [Google Scholar]
  7. Dolginova E. A., Roth E., Silman I., Weiner L. M. Chemical modification of Torpedo acetylcholinesterase by disulfides: appearance of a "molten globule" state. Biochemistry. 1992 Dec 8;31(48):12248–12254. doi: 10.1021/bi00163a039. [DOI] [PubMed] [Google Scholar]
  8. Emerson S. D., La Mar G. Solution structural characteristics of cyanometmyoglobin: resonance assignment of heme cavity residues by two-dimensional NMR. Biochemistry. 1990 Feb 13;29(6):1545–1556. doi: 10.1021/bi00458a028. [DOI] [PubMed] [Google Scholar]
  9. Esposito G., Lesk A. M., Molinari H., Motta A., Niccolai N., Pastore A. Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering. J Mol Biol. 1992 Apr 5;224(3):659–670. doi: 10.1016/0022-2836(92)90551-t. [DOI] [PubMed] [Google Scholar]
  10. Evans S. V., Brayer G. D. High-resolution study of the three-dimensional structure of horse heart metmyoglobin. J Mol Biol. 1990 Jun 20;213(4):885–897. doi: 10.1016/S0022-2836(05)80270-0. [DOI] [PubMed] [Google Scholar]
  11. Evans S. V., Brayer G. D. Horse heart metmyoglobin. A 2.8-A resolution three-dimensional structure determination. J Biol Chem. 1988 Mar 25;263(9):4263–4268. [PubMed] [Google Scholar]
  12. Feng Y. Q., Wand A. J., Sligar S. G. 1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562. Biochemistry. 1991 Aug 6;30(31):7711–7717. doi: 10.1021/bi00245a007. [DOI] [PubMed] [Google Scholar]
  13. Garcia-Moreno B., Chen L. X., March K. L., Gurd R. S., Gurd F. R. Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions. J Biol Chem. 1985 Nov 15;260(26):14070–14082. [PubMed] [Google Scholar]
  14. Griko Y. V., Privalov P. L., Venyaminov S. Y., Kutyshenko V. P. Thermodynamic study of the apomyoglobin structure. J Mol Biol. 1988 Jul 5;202(1):127–138. doi: 10.1016/0022-2836(88)90525-6. [DOI] [PubMed] [Google Scholar]
  15. Haynie D. T., Freire E. Structural energetics of the molten globule state. Proteins. 1993 Jun;16(2):115–140. doi: 10.1002/prot.340160202. [DOI] [PubMed] [Google Scholar]
  16. Hughson F. M., Baldwin R. L. Use of site-directed mutagenesis to destabilize native apomyoglobin relative to folding intermediates. Biochemistry. 1989 May 16;28(10):4415–4422. doi: 10.1021/bi00436a044. [DOI] [PubMed] [Google Scholar]
  17. Hughson F. M., Barrick D., Baldwin R. L. Probing the stability of a partly folded apomyoglobin intermediate by site-directed mutagenesis. Biochemistry. 1991 Apr 30;30(17):4113–4118. doi: 10.1021/bi00231a001. [DOI] [PubMed] [Google Scholar]
  18. Hughson F. M., Wright P. E., Baldwin R. L. Structural characterization of a partly folded apomyoglobin intermediate. Science. 1990 Sep 28;249(4976):1544–1548. doi: 10.1126/science.2218495. [DOI] [PubMed] [Google Scholar]
  19. Lecomte J. T., Cocco M. J. Structural features of the protoporphyrin-apomyoglobin complex: a proton NMR spectroscopy study. Biochemistry. 1990 Dec 18;29(50):11057–11067. doi: 10.1021/bi00502a007. [DOI] [PubMed] [Google Scholar]
  20. Macgregor R. B., Weber G. Estimation of the polarity of the protein interior by optical spectroscopy. Nature. 1986 Jan 2;319(6048):70–73. doi: 10.1038/319070a0. [DOI] [PubMed] [Google Scholar]
  21. Mann C. J., Matthews C. R. Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding. Biochemistry. 1993 May 25;32(20):5282–5290. doi: 10.1021/bi00071a002. [DOI] [PubMed] [Google Scholar]
  22. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  23. Matthews C. R. Pathways of protein folding. Annu Rev Biochem. 1993;62:653–683. doi: 10.1146/annurev.bi.62.070193.003253. [DOI] [PubMed] [Google Scholar]
  24. Mayer A., Ogawa S., Shulman R. G., Yamane T., Cavaleiro J. A., Rocha Gonsalves A. M., Kenner G. W., Smith K. M. Assignments of the paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin by selective deuteration. J Mol Biol. 1974 Jul 15;86(4):749–756. doi: 10.1016/0022-2836(74)90351-9. [DOI] [PubMed] [Google Scholar]
  25. Moore C. D., Lecomte J. T. Structural properties of apocytochrome b5: presence of a stable native core. Biochemistry. 1990 Feb 27;29(8):1984–1989. doi: 10.1021/bi00460a004. [DOI] [PubMed] [Google Scholar]
  26. Petros A. M., Mueller L., Kopple K. D. NMR identification of protein surfaces using paramagnetic probes. Biochemistry. 1990 Oct 30;29(43):10041–10048. doi: 10.1021/bi00495a005. [DOI] [PubMed] [Google Scholar]
  27. Petros A. M., Neri P., Fesik S. W. Identification of solvent-exposed regions of an FK-506 analog, ascomycin, bound to FKBP using a paramagnetic probe. J Biomol NMR. 1992 Jan;2(1):11–18. doi: 10.1007/BF02192797. [DOI] [PubMed] [Google Scholar]
  28. Pinker R. J., Lin L., Rose G. D., Kallenbach N. R. Effects of alanine substitutions in alpha-helices of sperm whale myoglobin on protein stability. Protein Sci. 1993 Jul;2(7):1099–1105. doi: 10.1002/pro.5560020704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Presta L. G., Rose G. D. Helix signals in proteins. Science. 1988 Jun 17;240(4859):1632–1641. doi: 10.1126/science.2837824. [DOI] [PubMed] [Google Scholar]
  30. Ptitsyn O. B., Pain R. H., Semisotnov G. V., Zerovnik E., Razgulyaev O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 1990 Mar 12;262(1):20–24. doi: 10.1016/0014-5793(90)80143-7. [DOI] [PubMed] [Google Scholar]
  31. Rance M., Sørensen O. W., Bodenhausen G., Wagner G., Ernst R. R., Wüthrich K. Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun. 1983 Dec 16;117(2):479–485. doi: 10.1016/0006-291x(83)91225-1. [DOI] [PubMed] [Google Scholar]
  32. Richardson J. S., Richardson D. C. Amino acid preferences for specific locations at the ends of alpha helices. Science. 1988 Jun 17;240(4859):1648–1652. doi: 10.1126/science.3381086. [DOI] [PubMed] [Google Scholar]
  33. Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965 Sep;13(2):482–495. doi: 10.1016/s0022-2836(65)80111-5. [DOI] [PubMed] [Google Scholar]
  34. Tandon S., Horowitz P. M. Reversible folding of rhodanese. Presence of intermediate(s) at equilibrium. J Biol Chem. 1989 Jun 15;264(17):9859–9866. [PubMed] [Google Scholar]
  35. Tirado-Rives J., Jorgensen W. L. Molecular dynamics simulations of the unfolding of apomyoglobin in water. Biochemistry. 1993 Apr 27;32(16):4175–4184. doi: 10.1021/bi00067a004. [DOI] [PubMed] [Google Scholar]
  36. Weiner H. Interaction of a spin-labeled analog of nicotinamide-adenine dinucleotide with alcohol dehydrogenase. I. Synthesis, kinetics, and electron paramagnetic resonance studies. Biochemistry. 1969 Feb;8(2):526–533. doi: 10.1021/bi00830a011. [DOI] [PubMed] [Google Scholar]
  37. Wishart D. S., Sykes B. D., Richards F. M. Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J Mol Biol. 1991 Nov 20;222(2):311–333. doi: 10.1016/0022-2836(91)90214-q. [DOI] [PubMed] [Google Scholar]
  38. Yip Y. K., Waks M., Beychok S. Influence of prosthetic groups on protein folding and subunit assembly. I. Conformational differences between separated human alpha- and beta- globins. J Biol Chem. 1972 Nov 25;247(22):7237–7244. [PubMed] [Google Scholar]

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