![]() Nuclear magnetic resonance (NMR) spectroscopy has a wide range of applications, including the determination of protein structures, characterization of protein-ligand interactions, and detection of changes in protein conformations and dynamics upon ligand binding (Kay, 2016 Kim, Howell, Van Horn, Jeon, & Sanders, 2009 Kitevski-LeBlanc & Prosser, 2012 Liang & Tamm, 2016 Liu, Horst, Katritch, Stevens, & Wuthrich, 2012 Oxenoid & Chou, 2013, 2016Rosenzweig & Kay, 2016 Ye, Van Eps, Zimmer, Ernst, & Prosser, 2016 Zhuang et al., 2013). The high-resolution structure of the designed anesthetic-binding protein offers unprecedented atomistic details about possible sites for anesthetic-protein interactions that are essential to the understanding of molecular mechanisms of general anesthesia. The NMR structures and Autodock analysis suggest that the pocket with the most favorable amphipathic property for anesthetic binding is located between the W15 side chains at the center of the dimeric hydrophobic core, with the possibility of two additional minor binding sites between the F12 and F52 ring stacks of each monomer. This observation was confirmed by the quantitative analysis using the Modelfree approach and by the NMR relaxation dispersion measurements. Qualitative analysis of the protein dynamics by reduced spectral density mapping revealed exchange contributions to the relaxation at many residues in the helices. The site of the 元8M mutation, which was previously shown to increase the halothane binding affinity by approximately 3.5-fold, is not part of the hydrophobic core presumably involved in the anesthetic binding but shows an elevated transverse relaxation (R(2)) rate. ![]() In comparison, the axes of the two helices from the linker to the C-terminus (helices 2 and 2') are wider apart from each other, creating a lateral access pathway around K47 from the aqueous phase to the center of the designed hydrophobic core. The two helices from the N-terminus to the linker (helices 1 and 1') are associated with each other in the dimer by the side-chain ring stacking of F12 and W15 along the long hydrophobic core and by a nearly perfect stretch of hydrophobic interactions between the complementary pairs of L4, L11, L18, and L25, all of which are located at the heptad e position along the helix-helix dimer interface. Two monomers of the helix-turn-helix motif form an antiparallel dimer as originally designed, but the high-resolution structure exhibits an asymmetric quaternary arrangement of the four helices. Using high resolution NMR, we solved the structure (Protein Data Bank ID: 2I7U) of a prototypical dimeric four-alpha-helix bundle, (Aalpha(2)-L1M/元8M)(2,) with designed specific binding pockets for volatile anesthetics. The four-alpha-helix bundle mimics the transmembrane domain of the Cys-loop receptor family believed to be the protein target for general anesthetics. This mechanism may be universal to anesthetic action on neuronal proteins. Our results revealed a novel mechanism of an induced fit between anesthetic molecule and its protein target, with the direct consequence of protein dynamics changing on a global rather than a local scale. Quantitative dynamics analyses, including Modelfree analysis of the relaxation data and the Carr-Purcell-Meiboom-Gill transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange both near and remote from the binding site. Whereas halothane produces minor changes in the monomer structure, the quaternary arrangement of the dimer is shifted by about half a helical turn and twists relative to each other, which leads to the closure of the lateral access pathway to the hydrophobic core. Hydrophobic interactions with residues A44 and L18 also contribute to stabilizing the bound halothane. The high-solution NMR structure, with a backbone root mean-square deviation of 1.72 A (2JST), and the NMR binding measurements revealed that the primary halothane binding site is located between two side-chains of W15 from each monomer, different from the initially designed anesthetic binding sites. ![]() ![]() In this study, we determined the high-resolution NMR structure of (Aalpha(2)-L1M/元8M)(2) in the presence of halothane, a clinically used volatile anesthetic. The structural and dynamical analyses of (Aalpha(2)-L1M/元8M)(2) in the absence of anesthetics (another study) showed a highly dynamic antiparallel dimer with an asymmetric arrangement of the four helices and a lateral accessing pathway from the aqueous phase to the hydrophobic core. As a model of the protein targets for volatile anesthetics, the dimeric four-alpha-helix bundle, (Aalpha(2)-L1M/元8M)(2), was designed to contain a long hydrophobic core, enclosed by four amphipathic alpha-helices, for specific anesthetic binding. ![]()
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