Solid-state sup 13 C NMR study of tyrosine protonation in dark-adapted bacteriorhodopsin

Journal Article · Tue Jun 12 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00475a022· OSTI ID:6534332

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 ^[1]

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1. Massachusetts Institute of Technology, Cambridge (USA)

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 degrees 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 bR568 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).

• OSTI ID: 6534332
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:23; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BARYONS
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HYDROXY ACIDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MOLECULAR STRUCTURE
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PH VALUE
PIGMENTS
PROTEINS
PROTONS
RESONANCE
RHODOPSIN
SPECTRA
STABLE ISOTOPES
TRACER TECHNIQUES
TYROSINE