Dissecting the Chemistry of Nicotinic Receptor-Ligand Interactions with Infrared Difference Spectroscopy

The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm−1 indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm−1 are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm−1 indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm−1 to 1690 and 1620 cm−1, respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions. The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm−1 indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm−1 are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm−1 indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm−1 to 1690 and 1620 cm−1, respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions. nicotinic acetylcholine receptor acetylcholine Fourier transform infrared carbamylcholine tetramethylamine resting to desensitized desensitized to desensitized The binding of a signaling molecule to an integral membrane receptor is a key event in many biological processes, including cell growth, intercellular communication, sensory perception, etc. Understanding the chemistry of membrane receptor-ligand interactions is thus central to understanding many biological phenomena. Because membrane receptors are the targets of pharmaceutical products, membrane receptor-ligand interactions are also of particular interest to the pharmaceutical industry. Unfortunately, the modern physical methodologies commonly used to probe the structural features responsible for receptor-ligand interactions are still limited in their application to membrane-imbedded receptors. The chemical nature of receptor-ligand interactions at the post-synaptic membrane have been studied intensively for the nicotinic acetylcholine receptor (nAChR1) fromTorpedo. Each of the two acetylcholine (ACh) binding sites on the nAChR consists of two subsites, an esterophilic subsite that binds the ester functional group of ACh and an anionic subsite that binds the quaternary ammonium cation (1Michelson M.J. Zeimal E.V. Acetylcholine: An Approach to the Molecular Mechanisms of Action. Pergamon Press, Oxford1973Google Scholar, 2Luyten W.H. J. Neurosci. Res. 1986; 16: 51-73Crossref PubMed Scopus (16) Google Scholar). Sequence analysis, affinity labeling, and site-directed mutagenesis identify both aromatic and negatively charged residues in the anionic subsite that likely interact with the charged nitrogen of ACh via cation-π electron interactions and/or hydrogen bonding (3Peterson G.L. J. Neurosci. Res. 1989; 22: 488-503Crossref PubMed Scopus (5) Google Scholar, 4Changeux J.P. Galzi J.L. Devillers-Thiery A. Bertrand D. Q. Rev. Biophys. 1992; 25: 395-432Crossref PubMed Scopus (154) Google Scholar, 5Tomaselli G.F. McLaughlin J.T. Jurman M.E. Hawrot E. Yellen G. Biophys. J. 1991; 60: 721-727Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 6Aylwin M.L. White M.M. Mol. Pharmacol. 1994; 46: 1149-1155PubMed Google Scholar, 7Nowak M.W. Kearney P.C. Sampson J.R. Saks M.E. Labarca C.G. Silverman S.K. Zhong W. Thorson J. Abelson J.N. Davidson N. Schultz P.G. Dougherty D.A. Lester H.A. Science. 1995; 268: 439-442Crossref PubMed Scopus (212) Google Scholar, 8Kearney P.C. Nowak M.W. Zhong W. Silverman S.K. Lester H.A. Dougherty D.A. Mol. Pharmacol. 1996; 50: 1401-1412PubMed Google Scholar, 9Czajkowski C. Kaufmann C. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6285-6289Crossref PubMed Scopus (106) Google Scholar). The crystal structure of the homologous ACh binding protein confirms the essential role for tyrosine and tryptophan residues in the anionic subsite (10Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1564) Google Scholar). In contrast, the nature of the chemical interactions that occur between ACh and the esterophilic subsite remain poorly understood. Infrared difference spectroscopy is a technique that has been used extensively to probe the subtle changes in chemical structure and/or local environment that are associated with protein conformational change (reviewed in Ref. 11Zscherp C. Barth A. Biochemistry. 2001; 40: 1875-1883Crossref PubMed Scopus (109) Google Scholar). The difference between spectra of the nAChR recorded in the presence and absence of the agonist carbamylcholine (Carb) exhibits a complex pattern of positive and negative bands that reflects shifts in the intensities and/or frequencies of vibrations from those amino acid residues whose structures are altered upon Carb binding (12Baenziger J.E. Miller K.W. Rothschild K.J. Biophys. J. 1992; 61: 983-992Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 13Baenziger J.E. Miller K.W. Rothschild K.J. Biochemistry. 1993; 32: 5448-5454Crossref PubMed Scopus (64) Google Scholar). Difference spectroscopy has been used to map the conformational states of the nAChR stabilized by both a variety of ligands and upon reconstitution into lipid bilayers of varying lipid compositions (14Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar, 15Ryan S.E. Blanton M.P. Baenziger J.E. J. Biol. Chem. 2001; 276: 4796-4803Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 16Ryan S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Baenziger J.E. Morris M.-L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The difference spectra also contain vibrational information pertaining to the physical interactions that occur between Carb and nAChR binding site residues. Extensive band overlap, however, has thus far limited interpretation of the latter in terms of either the types of amino acid side chains involved or the precise nature of the physical interactions that occur between protein side chains and Carb. In this report we present the first analysis of nAChR-ligand interactions as studied by infrared difference spectroscopy. To circumvent the problems of extensive band overlap, we developed a new “double ligand difference” method that can be used to isolate the vibrational features from protein side chains that interact directly with individual functional groups on a small molecule ligand. This new double ligand approach allows one to map individual ligand-receptor contacts and simplifies the difference spectra allowing for a more detailed interpretation of the data. We show here that the frequencies of two main protein vibrations are altered when the nAChR with the carbonyl of Carb. The frequencies of the vibrational bands suggest that a side in the esterophilic possibly an a hydrogen bond with the ester The results provide new insight into the nature of nAChR-ligand interactions and demonstrate the potential of infrared difference spectroscopy for dissecting the physical interactions that occur between a membrane receptor and ligand. The nAChR from Torpedo was on a and into of either or M.P. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). lipid compositions the nAChR in a that the membrane and S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). spectra recorded using the technique on either an or an both with a of the cell used to the spectra is in Carb difference two spectra of the resting nAChR recorded and the nAChR present on the of a The was to an one Carb a was recorded of the desensitized state. The between both the two resting spectra and the resting and desensitized spectra and the to the Carb. a to Carb from the the was many Each was recorded at using The spectra are of difference spectra recorded from at two difference spectra between and cm−1 and to an of difference spectra recorded as that the of Carb. Carb difference spectra recorded as for the Carb difference that the and both that the nAChR was with and thus the in the desensitized state. The double ligand difference spectra recorded as for the Carb difference spectra that one ligand Carb or was in the the ligand or was in both the and In the difference for the Carb and both the and Two spectra of the nAChR with to the site The was to the Carb to from the binding The between both the two spectra recorded with and the spectra recorded with first and Carb to the nAChR and and the was many the the both Carb and and the the the both ACh and and the the the both ACh and and the The difference between spectra of the nAChR recorded in the presence and absence of the agonist Carb to as a Carb difference exhibits a pattern of positive and negative bands that is in difference spectra recorded from nAChR J.E. Miller K.W. Rothschild K.J. Biochemistry. 1993; 32: 5448-5454Crossref PubMed Scopus (64) Google Scholar). bands reflect vibrations of Carb to the vibrational changes in the nAChR that occur upon the formation of physical interactions, as hydrogen cation-π electron interactions, between Carb and binding site and vibrational changes in the nAChR that from the conformational To the Carb difference bands first assigned to of the to Carb by band frequencies in the difference with those in spectra of Carb in as as by of difference and spectra recorded using ACh and an J.E. Miller K.W. Rothschild K.J. Biochemistry. 1993; 32: 5448-5454Crossref PubMed Scopus (64) Google Scholar). that reflect the structural changes associated with the conformational change by Carb difference spectra from nAChR that in the desensitized to Carb to as a Carb difference The Carb difference spectra positive intensity centered near 1655, 1547, 1430, and 1059 cm−1 that is in the Carb difference (14Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar, 15Ryan S.E. Blanton M.P. Baenziger J.E. J. Biol. Chem. 2001; 276: 4796-4803Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 16Ryan S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Baenziger J.E. Morris M.-L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). positive bands in the Carb difference thus reflect the main vibrational changes associated with of the nAChR from a resting to the desensitized in In contrast, bands that are by and that are to Carb reflect vibrational changes in protein residues that occur upon the formation of physical interactions between Carb and the desensitized features are in The of bands to either Carb or the conformational the that the Carb difference vibrational information the nature of interactions. vibrational features are at frequencies with the frequencies of side chains to be in the binding site The is a relatively intense band near 1516 cm−1 that is of tyrosine A. Biophys. Mol. Biol. 2000; PubMed Scopus Google Scholar). The intensity of the band is of a with mutagenesis that the of in agonist binding G.F. McLaughlin J.T. Jurman M.E. Hawrot E. Yellen G. Biophys. J. 1991; 60: 721-727Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 6Aylwin M.L. White M.M. Mol. Pharmacol. 1994; 46: 1149-1155PubMed Google Scholar). In bands are in of the associated with the vibrations of tryptophan and and cm−1 and between 1300 and 1400 residues. of near 1620 cm−1, could reflect interactions between Carb and additional binding site residues A. Biophys. Mol. Biol. 2000; PubMed Scopus Google Scholar). The difference spectra thus suggest a of interactions between Carb and the To vibrational features reflect interactions that occur between binding site residues and the quaternary amine of the difference between spectra recorded in the presence and absence of the agonist the functional was from in The difference exhibits many of the vibrational features in the Carb difference including bands near 1580, 1516, 1455, 1334, and between 1300 and 1400 are to the aromatic side chains of tyrosine and tryptophan and the side chains of In of the bands in the Carb difference that are to vibrational changes in residues with Carb are also present in the difference This that tyrosine and possibly both tryptophan and residues interact in with the quaternary The also that the interactions between the nAChR and Carb are by interactions with the quaternary amine functional The difference also exhibits positive intensity at of the frequencies centered near 1655, 1547, 1430, and 1059 cm−1 that as of the conformational The positive intensity at that to leads to a of the nAChR from a resting to a desensitized state. The of to the nAChR is that is an agonist of the receptor J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M.E. White M.M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The of was also in a of the interactions between the and the E. The difference between spectra of the nAChR recorded in the presence and absence of is thus to as a difference In to the band intensity are near and cm−1 and between both 1690 and 1668 cm−1 and and The reflect the vibrational frequencies and/or intensities of Carb. The latter reflect in the two ligands interact with the bands in the Carb difference that are in the difference can be assigned to the vibrational changes that occur as a of interactions between the of Carb and residues in the esterophilic interpretation of both the Carb and difference spectra in terms of both the of the amino involved and the nature of the individual contacts is by the extensive band for of many individual difference bands that as of amino acid side The in of individual bands that occur upon to are of side are also in the band shifts that occur in Carb difference spectra recorded J.E. Chew J.P. Biochemistry. PubMed Scopus Google Scholar). In the individual shifts in that occur upon Carb binding to the are directly to the nature of be of the To the difference and thus a interpretation of the we a novel double ligand difference method in The was to the difference between spectra of the nAChR recorded with and Carb to the binding Because both Carb and the nAChR in a desensitized the difference between spectra of the nAChR recorded with either or Carb to the binding sites to as a difference vibrational bands the conformational The vibrational features from the formation of physical interactions between the quaternary amine of Carb and the anionic subsite also be from the difference interactions are in the state. In the difference positive and negative vibrational bands to the vibrations of Carb and respectively, as as bands from esterophilic subsite residues whose structures and/or are altered upon with the of Carb the difference exhibits difference bands with either the Carb or difference spectra positive and negative bands to Carb and are the In contrast, relatively intense protein vibrations are in the cm−1 near and 1605 Other bands are to at the of The of protein vibrational features to and carboxylic acid etc. confirms that the of the physical contacts that occur between Carb and the nAChR at the anionic This the of the quaternary amine in agonist binding. Two of protein vibrations near cm−1 and cm−1, as as a positive near cm−1 reflect vibrational changes in esterophilic subsite residues that occur upon with the of Carb. To the functional group on Carb that with the esterophilic subsite residues to the band we recorded a of double ligand difference spectra using and/or as ligands The difference is to the difference the difference at and 1605 The of the difference spectra that both Carb and ACh interact with the nAChR at the esterophilic subsite in a In the group of Carb to physical contacts with residues in the esterophilic binding and is involved in the physical interactions that to the vibrational shifts at and 1605 the and also the two positive and negative difference at cm−1 and be a subtle change in intensity near The bands in the double ligand difference spectra near cm−1 and cm−1 thus be to interactions between esterophilic subsite residues and either the or ester of The main vibrational changes in the double ligand difference spectra thus reflect interactions between esterophilic subsite residues and the carbonyl of of the bands in the double ligand difference spectra is the frequencies of the vibrations suggest residues for mutagenesis The main vibrations in the double ligand difference spectra occur in the to and of the infrared and could reflect vibrational changes from the formation of hydrogen with carbonyl possibly a that the formation of a hydrogen bond between and the carbonyl the absence of band shifts in the and of the side chains are also in the ACh binding of the homologous ACh binding protein that could interact with the carbonyl of Carb (10Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1564) Google the precise of agonist binding to the site has been residues in the ACh binding protein tyrosine arginine and The nAChR residues are tyrosine and and tyrosine and arginine tyrosine and and and M. M. M. S. S. Nature. PubMed Scopus Google Scholar). The side chains of and vibrational intensity in the cm−1 of the infrared and thus are likely responsible for the vibrations in the difference A. Biophys. Mol. Biol. 2000; PubMed Scopus Google Scholar). In contrast, the and vibrations of arginine are near and cm−1, frequencies with the two of positive and negative near cm−1 and are a interpretation of is that the ester carbonyl of Carb with an arginine side when binding to the desensitized of the however, that the binding affinity of the ACh binding protein for ACh is A.B. D. van J. J. van van K. Sixma T.K. Nature. 2001; 411: PubMed Scopus Google Scholar). In contrast, the desensitized nAChR binds ACh with an affinity of Biochemistry. PubMed Scopus Google conformational between the binding sites of the ACh binding protein and the desensitized residues thus be in vibrational band frequencies are upon a change in of hydrogen a an increase in hydrogen bond leads to a in vibrational to the of Carb with the esterophilic subsite leads to shifts from 1668 and 1605 cm−1 to 1690 and 1620 cm−1, This could either the formation of a hydrogen bond with the ester carbonyl of Carb that is that when the esterophilic subsite is or the of a upon Carb binding. The interpretation of the that the nAChR has a affinity for Carb the shifts to reflect more complex vibrational are to the nature of the band shifts that occur upon the binding of Carb to the esterophilic difference spectroscopy a map of the vibrational changes that occur in the nAChR upon Carb binding. This map has been used extensively to the conformational states of the nAChR stabilized by a variety of ligands and upon reconstitution of the receptor into lipid bilayers of varying lipid compositions (14Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar, 15Ryan S.E. Blanton M.P. Baenziger J.E. J. Biol. Chem. 2001; 276: 4796-4803Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 16Ryan S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17Baenziger J.E. Morris M.-L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). we demonstrate that features in the Carb difference also provide a vibrational map of the physical interactions that occur between the nAChR and Carb. The difference exhibits bands at frequencies to and carboxylic acid are to a role in agonist binding protein vibrations suggest additional to interactions. We have developed a novel double ligand difference method that can be used to isolate the vibrational features from those residues in a integral membrane receptor that interact either directly or with functional groups on a small molecule ligand. The difference between spectra of the nAChR recorded with either Carb or to the binding sites exhibits protein vibrations from those residues in the esterophilic subsite that interact with the of Carb double ligand difference spectra show that vibrations reflect residues that interact either directly or with the ester carbonyl functional Two main band shifts from 1690 and 1620 cm−1 to 1668 and 1605 cm−1, respectively, are with the formation of a hydrogen bond between the ester carbonyl of Carb and an arginine side the of the double ligand difference method for dissecting the physical interactions that occur between a integral membrane receptor and a ligand. The of the double ligand difference spectra is from a in the Carb difference interpretation of the in terms of both the of the amino acid side chains that are involved in Carb binding and the nature of the In the two main band shifts from 1690 and 1620 cm−1 to 1668 and 1605 cm−1, respectively, that occur upon of the ester carbonyl of Carb with the esterophilic subsite are in either the Carb or difference The of band in the double ligand difference a precise of the band frequencies of the protein side chains that interact with the ester carbonyl in both the Carb and The of the the that difference spectra recorded and at to a of the protein vibrations here to an arginine residue. The main the nature of interactions from this are the The show that the main physical interactions between Carb and the nAChR from interactions between the quaternary amine and the anionic This is on the that the of features in the Carb difference to vibrational changes in residues that interact with Carb are also in the difference the and show that is of the nAChR in the desensitized The of the quaternary amine in both interactions and conformational change is in with show that is an agonist of the receptor J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M.E. White M.M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The difference spectra provide that the quaternary amine of Carb with tyrosine residues in the Carb binding to the nAChR leads to an increase in the vibrational intensity of a band near 1516 cm−1 indicating a change in the structure and/or local environment one or more tyrosine residues. This increase in tyrosine vibrational intensity is upon Carb binding to a desensitized nAChR that reflects physical interactions as to a vibrational associated with the conformational The increase in intensity near 1516 cm−1 is also upon binding the of the band to one or more interactions between one or more and the quaternary amine functional The intensity of the 1516 cm−1 band a key role for in agonist binding in with extensive using both chemical and site-directed mutagenesis J.P. Galzi J.L. Devillers-Thiery A. Bertrand D. Q. Rev. Biophys. 1992; 25: 395-432Crossref PubMed Scopus (154) Google Scholar, 5Tomaselli G.F. McLaughlin J.T. Jurman M.E. Hawrot E. Yellen G. Biophys. J. 1991; 60: 721-727Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 6Aylwin M.L. White M.M. Mol. Pharmacol. 1994; 46: 1149-1155PubMed Google Scholar, 7Nowak M.W. Kearney P.C. Sampson J.R. Saks M.E. Labarca C.G. Silverman S.K. Zhong W. Thorson J. Abelson J.N. Davidson N. Schultz P.G. Dougherty D.A. Lester H.A. Science. 1995; 268: 439-442Crossref PubMed Scopus (212) Google Scholar, 8Kearney P.C. Nowak M.W. Zhong W. Silverman S.K. Lester H.A. Dougherty D.A. Mol. Pharmacol. 1996; 50: 1401-1412PubMed Google Scholar, J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M.E. White M.M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). Vibrational features that are of the interactions between the quaternary amine and amino acid side chains are that a of residues to the anionic to tryptophan and the groups of and/or reflect changes in structure and/or local environment both types of residues upon with as has been C. Kaufmann C. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6285-6289Crossref PubMed Scopus (106) Google Scholar, W. J.P. Lester H.A. Dougherty D.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Other vibrational near 1620 cm−1, suggest the involvement of additional amino acid side positive and negative bands near cm−1 in the the the and difference spectra suggest the formation of a hydrogen bond between an esterophilic subsite and the ester carbonyl of Carb the nAChR is in the desensitized state. band are the frequencies are with an arginine side directly with the ester An arginine is likely to the agonist binding (10Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1564) Google Scholar). The of the double ligand difference technique to ligand-receptor contacts the potential of the technique for subtle structural that between the binding sites of homologous ligand binding difference spectra recorded from both the nAChR and the homologous ACh binding protein and/or in amino acid side chains in the esterophilic that the crystal structure of the ACh binding protein has been information could for the binding site of the approach could also be used to the structural for the nAChR receptor The approach used here is to The main is the to a membrane that to the of an in the presence of In biological to also K. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. 1996; 50: Scopus Google Scholar). variety of and one to the approach for a application J.E. Ryan S.E. M. Vibrational of and Scholar). of difference spectra are developed for (reviewed in Ref. 11Zscherp C. Barth A. Biochemistry. 2001; 40: 1875-1883Crossref PubMed Scopus (109) Google Scholar). The here be to a variety of both membrane-imbedded and to for the

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