Cation-Binding Location and Hydrogen-Exchange Sites for Gramicidin in SDS Micelles Using NOESY NMR

Hinton, James F.

doi:10.1006/jmrb.1996.0105

Cited by 11 publications

(5 citation statements)

References 7 publications

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“…In Gly 15 -GA, the Trp 11 side chain is oriented away from the channel axis, as shown in Figure . The cation binding site for GA has been identified as a “pocket” formed by residues 10−15 ( 24 , − . The binding of a cation in this “pocket” involves the carbonyl oxygen atoms of the amino acid residues and the long-range ion−tryptophan dipole interactions.…”

Section: Resultsmentioning

confidence: 99%

The Structure, Cation Binding, Transport, and Conductance of Gly₁₅-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles^,

et al. 2003

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To further investigate the effect of single amino acid substitution on the structure and function of the gramicidin channel, an analogue of gramicidin A (GA) has been synthesized in which Trp(15) is replaced by Gly in the critical aqueous interface and cation binding region. The structure of Gly(15)-GA incorporated into SDS micelles has been determined using a combination of 2D-NMR spectroscopy and molecular modeling. Like the parent GA, Gly(15)-GA forms a dimeric channel composed of two single-stranded, right-handed beta(6.3)-helices joined by hydrogen bonds between their N-termini. The replacement of Trp(15) by Gly does not have a significant effect on backbone structure or side chain conformations with the exception of Trp(11) in which the indole ring is rotated away from the channel axis. Measurement of the equilibrium binding constants and Delta G for the binding of monovalent cations to GA and Gly(15)-GA channels incorporated into PC vesicles using (205)Tl NMR spectroscopy shows that monovalent cations bind much more weakly to the Gly(15)-GA channel entrance than to GA channels. Utilizing the magnetization inversion transfer NMR technique, the transport of Na(+) ions through GA and Gly(15)-GA channels incorporated into PC/PG vesicles has been investigated. The Gly(15) substitution produces an increase in the activation enthalpy of transport and thus a significant decrease in the transport rate of the Na(+) ion is observed. The single-channel appearances show that the conducting channels have a single, well-defined structure. Consistent with the NMR results, the single-channel conductances are reduced by 30% and the lifetimes by 70%. It is concluded that the decrease in cation binding, transport, and conductance in Gly(15)-GA results from the removal of the Trp(15) dipole and, to a lesser extent, the change in orientation of Trp(11).

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Section: Resultsmentioning

confidence: 99%

The Structure, Cation Binding, Transport, and Conductance of Gly₁₅-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles^,

et al. 2003

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“…The vertical arrows indicate the frequencies of the selective saturation by the hyperbolic secant-shaped pulse train. Clearly, with careful adjustment of pH to avoid base-catalyzed exchange with water (Arseniev et al, 1985;Hinton, 1996), saturation is well localized to the protons selected. Although high 1 H resolution is easily achievable in SDS, intermolecular NOE build-up is more favorable in a lipid bilayer environment.…”

Section: Resultsmentioning

confidence: 99%

“…As a simplified model, the well-characterized gramicidin A channel (HCO-L-Val 1 -Gly 2 -L-Ala 3 -D-Leu 4 -L-Ala 5 -D-Val 6 -L-Val 7 -D-Val 8 -L-Trp 9 -D-Leu 10 -L-Trp 11 -D-Leu 12 -L-Trp 13 -D-Leu 14 -L-Trp 15 -NHCH 2 CH 2 OH) offers at least the following four advantages, which are unavailable in many other systems: 1) high achievable nuclear magnetic resonance (NMR) spectral resolution of the peptide permits absolute identification of individual amino acid residues along the transmembrane channel (Arseniev et al, 1985;Cross, 1994;Hinton, 1996), thus allowing direct quantitation of the sitespecific interaction between anesthetics and the channel peptide; 2) a spectroscopically resolved protein-lipid interface allows testing of popular hypotheses relating to the protein-lipid interaction; 3) an environment that is unique to membrane proteins but not to soluble proteins can be unambiguously defined at the interfacial region, where protein, lipid, and water come into contact; and 4) because gramicidin forms a functional channel with resolved threedimensional structures, it provides a unique system for determining the structural and functional consequences of anesthetic interaction.…”

Section: Introductionmentioning

confidence: 99%

Distinctly Different Interactions of Anesthetic and Nonimmobilizer with Transmembrane Channel Peptides

Tang

Liachenko

et al. 1999

Biophysical Journal

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Although it plays no clinical role in general anesthesia, gramicidin A, a transmembrane channel peptide, provides an excellent model for studying the specific interaction between volatile anesthetics and membrane proteins at the molecular level. We show here that a pair of structurally similar volatile anesthetic and nonimmobilizer (nonanesthetic), 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1, 2-dichlorohexafluorocyclobutane (F6), respectively, interacts differently with the transmembrane peptide. With 400 microM gramicidin A in a vesicle suspension of 60 mM phosphatidylcholine-phosphatidylglycerol (PC/PG), the intermolecular cross-relaxation rate constants between (19)F of F3 and (1)H in the chemical shift regions for the indole and backbone amide protons were 0.0106 +/- 0.0007 (n = 12) and 0.0105 +/- 0.0014 (n = 8) s(-1), respectively. No cross-relaxation was measurable between (19)F of F6 and protons in these regions. Sodium transport study showed that with 75 microM gramicidin A in a vesicle suspension of 66 mM PC/PG, F3 increased the (23)Na apparent efflux rate constant from 149.7 +/- 7.2 of control (n = 3) to 191.7 +/- 12.2 s(-1) (n = 3), and the apparent influx rate constant from 182.1 +/- 15.4 to 222.8 +/- 21.7 s(-1) (n = 3). In contrast, F6 had no effects on either influx or efflux rate. It is concluded that the ability of general anesthetics to interact with amphipathic residues near the peptide-lipid-water interface and the inability of nonimmobilizer to do the same may represent some characteristics of anesthetic-protein interaction that are of importance to general anesthesia.

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“…One would expect that if a Trp residue existed near the channel entrance (at the membrane/aqueous interface-where interaction with bulk water seems more likely), it is possible that the hydrophilic region (the indole-NH) will be able to hydrogen-bond to external H 2 O, whereas its nonpolar surface remains buried in the membrane. Previously published data indicate that Trp 9 and Trp 11 of native gA do not supply any sites available for solvent exchange, suggesting that these two Trp residues exist in the more hydrophobic portion of the micelle (Hinton, 1996). It is likely, then, that Trp residues more near the aqueous/ membrane interface (Trp 15 and Trp 13 ) that interact with bulk water would enhance channel lifetime by possessing some hydrophilic properties, a conclusion supported by the incorporation data from Easton (1989).…”

Section: The Effects Of Trp!phe Replacements On Incorporation and Channel Lifetimementioning

confidence: 98%

Effects of Phenylalanine Substitutions in Gramicidin A on the Kinetics of Channel Formation in Vesicles and Channel Structure in SDS Micelles

Jordan

Easton

Hinton

2005

Biophysical Journal

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The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.

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Cation-Binding Location and Hydrogen-Exchange Sites for Gramicidin in SDS Micelles Using NOESY NMR

Cited by 11 publications

References 7 publications

The Structure, Cation Binding, Transport, and Conductance of Gly₁₅-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles^,

The Structure, Cation Binding, Transport, and Conductance of Gly₁₅-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles^,

Distinctly Different Interactions of Anesthetic and Nonimmobilizer with Transmembrane Channel Peptides

Effects of Phenylalanine Substitutions in Gramicidin A on the Kinetics of Channel Formation in Vesicles and Channel Structure in SDS Micelles

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Cation-Binding Location and Hydrogen-Exchange Sites for Gramicidin in SDS Micelles Using NOESY NMR

Cited by 11 publications

References 7 publications

The Structure, Cation Binding, Transport, and Conductance of Gly15-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles,

The Structure, Cation Binding, Transport, and Conductance of Gly15-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles,

Distinctly Different Interactions of Anesthetic and Nonimmobilizer with Transmembrane Channel Peptides

Effects of Phenylalanine Substitutions in Gramicidin A on the Kinetics of Channel Formation in Vesicles and Channel Structure in SDS Micelles

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The Structure, Cation Binding, Transport, and Conductance of Gly₁₅-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles^,

The Structure, Cation Binding, Transport, and Conductance of Gly₁₅-Gramicidin A Incorporated into SDS Micelles and PC/PG Vesicles^,