Summary
The deuteration of the tryptophan residues of hen egg white lysozyme, bovineα-lactalbumin and bovineβ-lactoglobulin in d-TFA has been studied by PMR spectroscopy. It is found that short times of exposure to d-TFA allow selective deuteration at the C-2 position with only a small amount of deuteration at the C-5 position, as expected from studies on model peptides described in the previous paper. The proteins studied essentially regained their native structures after the treatment, except for broadening and shifting of the histidine resonances in the case ofα-lactalbumin. Selective deuteration at the tryptophan C-2 position was readily observed by difference spectroscopy of the denatured protein, but PMR difference spectra of the same proteins in benign solvents did not contain resonances from all of the exchanged protons. Some resonances could not be observed because of line broadening, which causes the resonances to fall below the limit of sensitivity of detection at 100 MHz. Deuteration by brief exposure to d-TFA should be useful for the identification of tryptophan resonances in the PMR spectra of native proteins.
The deuteration of all the aromatic protons of tryptophan residues in proteins by immersion in d-TFA for 4 hours at room temperature was studied. This technique is unlikely to be of general use for the simplification of the aromatic region of the PMR spectra of native proteins because of the degradation of tryptophan residues which results from the acid treatment.
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Norton, R. S. and Bradbury, J. H., 1976. Mol. Cell. Biochem. preceding paper in this issue.
Bak, B., Led, J. J. and Pedersen, E. J., 1969. Acta Chem. Scand. 23, 3051–3054.
Bak, B., Led, J. J. and Pedersen, E. J., 1969. J. Mol. Spect. 32, 151–156.
Holt, L. A., Milligan, B. and Rivett, D. E., 1971. Biochemistry 10, 3559–3564.
Holt, L. A. and Milligan, B., 1973. Aust. J. Biol. Sci. 26, 871–876.
King, N. L. R. and Bradbury, J. H., 1971. Nature 229, 404–406.
Bradbury, J. H. and King, N. L. R., 1971. Aust. J. Chem. 24, 1703–1714.
Barman, T. E., 1970. Biochim. Biophys. Acta 214, 242–244.
Shugar, D., 1952. Biochim. Biophys. Acta 8, 302–309.
Bradbury, J. H. and Norton, R. S., 1975. Eur. J. Biochem. 53, 387–396.
Marier, J. R. and Rose, D., 1964. Anal. Biochem 7, 304–314.
McDonald, C. C. and Phillips, W. D., 1967. J. Amer. Chem. Soc. 89, 6332–6341.
Sternlicht, H. and Wilson, D., 1967. Biochemistry 6, 2881–2892.
Bradbury, J. H. and Norton, R. S., 1974. Int. J. Peptide Protein Res. 6, 295–302.
Campbell, I. D., Dobson, C. M., Williams, R. J. P. and Xavier, A. V., 1973. Ann. N.Y. Acad. Sci. 222, 163–174.
Bradbury, J. H. and Brown, L. R., 1973. Eur. J. Biochem. 40, 565–576.
Campbell, I. D., Dobson, C. M., and Williams, R. J. P., 1975. Proc. Roy. Soc. Lond. B 189, 485–502.
Norton, R. S., Ph.D. Thesis, Australian National University, 1974.
Hayashi, K., Imoto, T., Funatsu, G., and Funatsu, M., 1965. J Biochem. (Tokyo) 58, 227–235.
Oldfield, E., Norton, R. S. and Allerhand, A., 1975. J. Biol. Chem. 250, 6381–6402.
Cohen, J. S. and Jardetzky, O., 1968. Proc. Natl. Acad. Sci. U.S.A. 60, 92–99.
Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C. and Rupley, J. A., 1972. in “The Enzymes“ (Boyer, P. D., ed.) Vol. VII, 3rd ed., pp. 665–868, Academic Press, New York.
Brew, K., Castellino, F. J., Vanaman, T. C. and Hill, R. L., 1970. J. Biol. Chem. 245, 4570–4582.
Bradbury, J. H. and King, N. L. R., 1969. Aust. J. Chem. 22, 1083–1089.
McKenzie, H. A. and Sawyer, W. H., 1967. Nature 214, 1101–1104.
Timasheff, S. N., Townend, R., and Mescanti, L., 1966. J. Biol. Chem. 241, 1863–1870.
Uphaus, R. A., Grossweiner, L. I., Katz, J. J. and Kopple, K. D., 1959. Science 129, 641–643.
Bradbury, J. H., 1973. Adv. Prot. Chem. 27, 111–211.
Tanford, C., 1968. Adv. Prot. Chem. 23, 121–282.
Bradbury, J. H., Chapman, B. E. and King, N. L. R., 1971. Int. J. Protein Res. 3, 351–356.
Kubo, R. and Tomita, K., 1954. J. Phys. Soc. Japan 9, 888–919.
Glickson, J. D., Phillips, W. D. and Rupley, J. A., 1971. J. Amer. Chem. Soc. 93, 4031–4038.
Dubin, S. B., Clark, N. A. and Benedek, G. B., 1971. J. Chem. Phys. 54, 5158–5164.
Cozzone, P. J., Opella, S. J., Jardetzky, O., Berthou, J. and Jolles, P., 1975. Proc. Natl. Acad. Sci., U.S. 72, 2095–2098.
Wilbur, D. J., Norton, R. S., Clouse, A. O., Addleman, R. and Allerhand, A., 1976. J. Amer. Chem. Soc., in press.
Rawitch, A. B., 1972. Arch. Biochem. Biophys. 151, 22–27.
Townend, R., Herskovits, T. T., Timasheff, S. N. and Gorbunoff, M. J., 1969. Arch. Biochem. Biophys. 129, 567–580.
Seibles, T. S., 1969. Biochemistry 8, 2949–2954.
Campbell, I. D., Dobson, C. M., Williams, R. J. P. and Xavier, A. V., 1973. J. Magn. Res. 11, 172–181.
Campbell, I. D., Dobson, C. M. and Williams, R. J. P., 1974. J. Chem. Soc. (Chem. Commun.) 888–889.
Campbell, I. D., Dobson, C. M., Williams, R. J. P. and Wright, P. E., 1975. F.E.B.S. Letters 57, 96–99.
Campbell, I. D., Dobson, C. M. and Williams, R. J. P., 1975. Proc. Roy. Soc. Lond. B 189, 503–509.
Campbell, I. D., Dobson, C. M. and Williams, R. J. P., 1975. Proc. Roy. Soc. Lond. A 345, 41–59.
Dobson, C. M., Moore, G. R., and Williams, R. J. P., 1975. F.E.B.S. Letters 51, 60–65.
Bradbury, J. H. and Norton, R. S., 1973. Biochim. Biophys. Acta 328, 10–19.
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Bradbury, J.H., Norton, R.S. Proton magnetic resonance spectroscopic studies of proteins containing deuterated tryptophan residues. Mol Cell Biochem 13, 113–125 (1976). https://doi.org/10.1007/BF01837061
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DOI: https://doi.org/10.1007/BF01837061