Dynamics of amino acid side chains in membrane proteins by high field solid state deuterium nuclear magnetic resonance spectroscopy. Phenylalanine, tyrosine, and tryptophan.

R. A. Kinsey, A. Kintanar, E. Oldfield

Research output: Contribution to journalArticle

Abstract

We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all such motions are frozen out, i.e. occur at rates less than 10(4) s-1, and axially symmetric (eta approximately 0.05) line shapes are observed. At T greater than 91 degrees C, phenylalanine-, tyrosine-, and tryptophan-labeled membrane spectra undergo dramatic narrowing to an isotropic line of approximately 4-9 kHz width. This transition is a reflection of the loss of tertiary structure in the membrane protein with resultant fast unrestricted motion of these aromatic side chains, and is only partly reversible. These results, in conjunction with those obtained using [gamma-2H6]valine-labeled bacteriorhodopsin (Kinsey, R. A., Kintanar, A., Tsai, M-D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146-4149) indicate the rather rigid nature of amino acid side chains in the H. halobium purple membrane, the principal fast lage amplitude motions being methyl group rotation and discontinuous benzene ring "flipping."

Original languageEnglish (US)
Pages (from-to)9028-9036
Number of pages9
JournalJournal of Biological Chemistry
Volume256
Issue number17
StatePublished - Sep 10 1981
Externally publishedYes

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Deuterium
Phenylalanine
Tryptophan
Nuclear magnetic resonance spectroscopy
Tyrosine
Bacteriorhodopsins
Membrane Proteins
Magnetic Resonance Spectroscopy
Purple Membrane
Halobacterium salinarum
Membranes
Amino Acids
Nuclear magnetic resonance
Aromatic Amino Acids
Valine
Benzene
Labeling
Temperature
Tensors
Data acquisition

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Dynamics of amino acid side chains in membrane proteins by high field solid state deuterium nuclear magnetic resonance spectroscopy. Phenylalanine, tyrosine, and tryptophan.",
abstract = "We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all such motions are frozen out, i.e. occur at rates less than 10(4) s-1, and axially symmetric (eta approximately 0.05) line shapes are observed. At T greater than 91 degrees C, phenylalanine-, tyrosine-, and tryptophan-labeled membrane spectra undergo dramatic narrowing to an isotropic line of approximately 4-9 kHz width. This transition is a reflection of the loss of tertiary structure in the membrane protein with resultant fast unrestricted motion of these aromatic side chains, and is only partly reversible. These results, in conjunction with those obtained using [gamma-2H6]valine-labeled bacteriorhodopsin (Kinsey, R. A., Kintanar, A., Tsai, M-D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146-4149) indicate the rather rigid nature of amino acid side chains in the H. halobium purple membrane, the principal fast lage amplitude motions being methyl group rotation and discontinuous benzene ring {"}flipping.{"}",
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T1 - Dynamics of amino acid side chains in membrane proteins by high field solid state deuterium nuclear magnetic resonance spectroscopy. Phenylalanine, tyrosine, and tryptophan.

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N2 - We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all such motions are frozen out, i.e. occur at rates less than 10(4) s-1, and axially symmetric (eta approximately 0.05) line shapes are observed. At T greater than 91 degrees C, phenylalanine-, tyrosine-, and tryptophan-labeled membrane spectra undergo dramatic narrowing to an isotropic line of approximately 4-9 kHz width. This transition is a reflection of the loss of tertiary structure in the membrane protein with resultant fast unrestricted motion of these aromatic side chains, and is only partly reversible. These results, in conjunction with those obtained using [gamma-2H6]valine-labeled bacteriorhodopsin (Kinsey, R. A., Kintanar, A., Tsai, M-D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146-4149) indicate the rather rigid nature of amino acid side chains in the H. halobium purple membrane, the principal fast lage amplitude motions being methyl group rotation and discontinuous benzene ring "flipping."

AB - We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all such motions are frozen out, i.e. occur at rates less than 10(4) s-1, and axially symmetric (eta approximately 0.05) line shapes are observed. At T greater than 91 degrees C, phenylalanine-, tyrosine-, and tryptophan-labeled membrane spectra undergo dramatic narrowing to an isotropic line of approximately 4-9 kHz width. This transition is a reflection of the loss of tertiary structure in the membrane protein with resultant fast unrestricted motion of these aromatic side chains, and is only partly reversible. These results, in conjunction with those obtained using [gamma-2H6]valine-labeled bacteriorhodopsin (Kinsey, R. A., Kintanar, A., Tsai, M-D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146-4149) indicate the rather rigid nature of amino acid side chains in the H. halobium purple membrane, the principal fast lage amplitude motions being methyl group rotation and discontinuous benzene ring "flipping."

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