Fig. 4.  Nuclear magnetic resonance (NMR) spectroscopy to study sevoflurane binding sites in the lymphocyte function-associated antigen-1 (LFA-1) inserted (I) domain. (A ) Heteronuclear 1H, 15N single-quantum coherence-NMR spectroscopy was used to map sevoflurane-binding sites in the LFA-1 I domain. An overlay of two heteronuclear single-quantum coherence spectra obtained with 0 mM (blue ) and ∼ 9.9 mM sevoflurane (red ) is shown. The chemical shift of most resonances, such as Ser245 (inset ), remained unchanged (δppm less than 0.01), demonstrating that even at saturation, sevoflurane does not perturb the global fold (structure) of the I domain. A small subset of resonances, including Thr291 in the β6-α7 loop and Tyr307 in the C-terminal α7 helix, undergo large chemical shift changes (δppm greater than 0.05) upon the addition of sevoflurane. (B ) Structure of the I domain showing that amide nitrogen residues were affected (δppm greater than 0.05) by the addition of ∼9.9 mM sevoflurane. Gray  represents residues unperturbed by sevoflurane, whereas red  represents residues that met or exceeded the threshold for perturbation. α1 and α7 helices, as well as β1, β4, and β6 strands, are labeled. Residues Thr291, Ser245, and Tyr307 are arrowed  and labeled. The yellow spheres  represent the Mg2+ion at the metal ion-dependent adhesion site. The green arrow  denotes the cavity that contains the drug-binding sites for the αI allosteric antagonists and isoflurane. (C ) Scaled chemical shift perturbation of saturated sevoflurane mapped onto the LFA-1 I domain protein sequence and secondary structure. Chemical shift perturbation was calculated as [0.2(δNsev−δN0)2 + (δHsev−δH0)2]1/2. A subset of resonances (Asn163, Lys188, Arg221, Ile258, Ile259, Lys268, Phe299, Thr300, Ile306) was excluded from consideration because of overlap, uncertainty in the chemical shift assignment during titration, and/or exchange broadening during drug addition. Unperturbed Ser245 and perturbed Thr291 are indicated with arrows . Secondary structure assignments: β1 (130–137), α1 (144–160), β2 (166–173), β2′ (177–181), α2 (183–188), α3 (192–196), α4 (208–218), β3 (231–238), α5 (249–251), β4 (255–261), α6 (268–277), β5 (286–289), and α7 (293–305). For comparison, residues that shifted upon the addition of the LFA-1 allosteric inhibitor lovastatin are highlighted in yellow .

Fig. 4.  Nuclear magnetic resonance (NMR) spectroscopy to study sevoflurane binding sites in the lymphocyte function-associated antigen-1 (LFA-1) inserted (I) domain. (A ) Heteronuclear 1H, 15N single-quantum coherence-NMR spectroscopy was used to map sevoflurane-binding sites in the LFA-1 I domain. An overlay of two heteronuclear single-quantum coherence spectra obtained with 0 mM (blue ) and ∼ 9.9 mM sevoflurane (red ) is shown. The chemical shift of most resonances, such as Ser245 (inset ), remained unchanged (δppm less than 0.01), demonstrating that even at saturation, sevoflurane does not perturb the global fold (structure) of the I domain. A small subset of resonances, including Thr291 in the β6-α7 loop and Tyr307 in the C-terminal α7 helix, undergo large chemical shift changes (δppm greater than 0.05) upon the addition of sevoflurane. (B ) Structure of the I domain showing that amide nitrogen residues were affected (δppm greater than 0.05) by the addition of ∼9.9 mM sevoflurane. Gray  represents residues unperturbed by sevoflurane, whereas red  represents residues that met or exceeded the threshold for perturbation. α1 and α7 helices, as well as β1, β4, and β6 strands, are labeled. Residues Thr291, Ser245, and Tyr307 are arrowed  and labeled. The yellow spheres  represent the Mg2+ion at the metal ion-dependent adhesion site. The green arrow  denotes the cavity that contains the drug-binding sites for the αI allosteric antagonists and isoflurane. (C ) Scaled chemical shift perturbation of saturated sevoflurane mapped onto the LFA-1 I domain protein sequence and secondary structure. Chemical shift perturbation was calculated as [0.2(δNsev−δN0)2 + (δHsev−δH0)2]1/2. A subset of resonances (Asn163, Lys188, Arg221, Ile258, Ile259, Lys268, Phe299, Thr300, Ile306) was excluded from consideration because of overlap, uncertainty in the chemical shift assignment during titration, and/or exchange broadening during drug addition. Unperturbed Ser245 and perturbed Thr291 are indicated with arrows . Secondary structure assignments: β1 (130–137), α1 (144–160), β2 (166–173), β2′ (177–181), α2 (183–188), α3 (192–196), α4 (208–218), β3 (231–238), α5 (249–251), β4 (255–261), α6 (268–277), β5 (286–289), and α7 (293–305). For comparison, residues that shifted upon the addition of the LFA-1 allosteric inhibitor lovastatin are highlighted in yellow .

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