Mechanism of proton pumping in bacterioring psin by solid-state NMR: The protonation state of tyrosine in the light-adapted and M states

Journal Article · Tue Aug 27 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00098a013· OSTI ID:5602857

McDermott, A E; Thompson, L K; Griffin, R G ^[1]; Winkel, C; Lugtenburg, J ^[2]; Farrar, M R ^[3]; Pelletier, S; Herzfeld, J ^[4]

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1. Massachusetts Inst. of Tech., Cambridge (United States)
2. Rijksuniversiteit te Leiden (Netherlands)
3. Massachusetts Inst. of Tech., Cambridge (United States) Brandeis Univ., Waltham, MA (United States)
4. Brandeis Univ., Waltham, MA (United States)

Solid-state {sup 13}C NMR spectra were employed to characterize the ping nation state of tyrosine in the light-adapted (bR{sub 568}) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for (4{prime}-{sup 13}C)Tyr-labeled bR, regenerated with (14-{sup 13}C)retinal has distinct chemical shifts for bR{sub 555}, for bR{sub 569}, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate areing ily distinguished on the basis of the chemical shift of the 4{prime}-{sup 13}C label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, the authors show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at {minus}80{degrees}C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. These changes were reversible when the samping as warmed, although on a time scale slower than the relaxation of the retinal back to the bR{sub 568} conformer. Such small changes may be interpreted as alterations in the environment or the hydrogen bonding of some of the tyrosines, but not as evidence for the formation of tyrosinate. In summary, these data indicate that no stable tyrosinate is found in the dark-adapted, light-adapted, or M states of bacteriorhodopsin.

• OSTI ID: 5602857
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:34; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BARYONS
BIOCHEMICAL REACTION KINETICS
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACID ESTERS
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ESTERS
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HYDROXY ACIDS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOTOSYNTHETIC BACTERIA
PIGMENTS
PROTEINS
PROTONS
REACTION KINETICS
RESONANCE
RETINOIC ACID
RHODOPSIN
STABLE ISOTOPES
TYROSINE