Solid-State 13C NMR Study of Tyrosine Protonation in Dark-Adapted Bacteriorhodopsin

J. Herzfeld, S. K. Das Gupta, M. R. Farrar, G. S. Harbison, A. E. McDermott, S. L. Pelletier, D. P. Raleigh, S. O. Smith, C. Winkel, J. Lugtenburg, R. G. Griffin

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Abstract

Solid-state 13C MAS NMR spectra were obtained for dark-adapted bacteriorhodopsin (bR) labeled with [4′−13C]Tyr. Difference spectra (labeled minus natural abundance) taken at pH values between 2 and 12, and temperatures between 20 and −90 °C, exhibit a single signal centered at 156 ppm, indicating that the 11 tyrosines are protonated over a wide pH range. However, at pH 13, a second line appears in the spectrum with an isotropic shift of 165 ppm. Comparisons with solution and solid-state spectra of model compounds suggest that this second line is due to the formation of tyrosinate. Integrated intensities indicate that about half of the tyrosines are deprotonated at pH 13. This result demonstrates that deprotonated tyrosines in a membrane protein are detectable with solid-state NMR and that neither the bR568 nor the bR555 form of bR present in the dark-adapted state contains a tyrosinate at pH values between 2 and 12. Deprotonation of a single tyrosine in bR56S would account for 3.6% of the total tyrosine signal, which would be detectable with the current signal-to-noise ratio. We observe a slight heterogeneity and subtle line-width changes in the tyrosine signal between pH 7 and pH 12, which we interpret to be due to protein environmental effects (such as changes in hydrogen bonding) rather than complete deprotonation of tyrosine residue(s).

Original languageEnglish (US)
Pages (from-to)5567-5574
Number of pages8
JournalBiochemistry
Volume29
Issue number23
DOIs
StatePublished - Jun 1 1990

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Bacteriorhodopsins
Protonation
Tyrosine
Nuclear magnetic resonance
Deprotonation
Signal-To-Noise Ratio
Hydrogen Bonding
Carbon-13 Magnetic Resonance Spectroscopy
Linewidth
Environmental impact
Signal to noise ratio
Hydrogen bonds
Membrane Proteins
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Herzfeld, J., Das Gupta, S. K., Farrar, M. R., Harbison, G. S., McDermott, A. E., Pelletier, S. L., ... Griffin, R. G. (1990). Solid-State 13C NMR Study of Tyrosine Protonation in Dark-Adapted Bacteriorhodopsin. Biochemistry, 29(23), 5567-5574. https://doi.org/10.1021/bi00475a022

Solid-State 13C NMR Study of Tyrosine Protonation in Dark-Adapted Bacteriorhodopsin. / Herzfeld, J.; Das Gupta, S. K.; Farrar, M. R.; Harbison, G. S.; McDermott, A. E.; Pelletier, S. L.; Raleigh, D. P.; Smith, S. O.; Winkel, C.; Lugtenburg, J.; Griffin, R. G.

In: Biochemistry, Vol. 29, No. 23, 01.06.1990, p. 5567-5574.

Research output: Contribution to journalArticle

Herzfeld, J, Das Gupta, SK, Farrar, MR, Harbison, GS, McDermott, AE, Pelletier, SL, Raleigh, DP, Smith, SO, Winkel, C, Lugtenburg, J & Griffin, RG 1990, 'Solid-State 13C NMR Study of Tyrosine Protonation in Dark-Adapted Bacteriorhodopsin', Biochemistry, vol. 29, no. 23, pp. 5567-5574. https://doi.org/10.1021/bi00475a022
Herzfeld J, Das Gupta SK, Farrar MR, Harbison GS, McDermott AE, Pelletier SL et al. Solid-State 13C NMR Study of Tyrosine Protonation in Dark-Adapted Bacteriorhodopsin. Biochemistry. 1990 Jun 1;29(23):5567-5574. https://doi.org/10.1021/bi00475a022
Herzfeld, J. ; Das Gupta, S. K. ; Farrar, M. R. ; Harbison, G. S. ; McDermott, A. E. ; Pelletier, S. L. ; Raleigh, D. P. ; Smith, S. O. ; Winkel, C. ; Lugtenburg, J. ; Griffin, R. G. / Solid-State 13C NMR Study of Tyrosine Protonation in Dark-Adapted Bacteriorhodopsin. In: Biochemistry. 1990 ; Vol. 29, No. 23. pp. 5567-5574.
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