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The Substituted‐Cysteine Accessibility Method (SCAM) to Elucidate Membrane Protein Structure

George Liapakis¹, Merrill M. Simpson¹, Jonathan A. Javitch¹

¹Columbia University, New York, New York

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 4.15

DOI:

10.1002/0471142301.ns0415s08

Online Posting Date:

May, 2001

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Abstract

The substituted-cysteine accessibility method (SCAM) provides an approach to identifying the residues in the membrane-spanning segments that line a channel, transporter, or binding-site crevice. SCAM can also be used to determine differences in the structures of the membrane-spanning segments in different functional states of the proteins, to map electrostatic potential in the membrane-spanning domains, and to size a channel or binding-site crevice. The protocol in this unit describes the use of SCAM to map the binding-site crevice of a G-protein coupled receptor (GPCR) which binds ligand within the transmembrane portion of the receptor.

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Unit Introduction
Basic Protocol: Reaction of Sulfhydryl Reagents with Substituted-Cysteine Mutanats
Support Protocol: Protection of Substituted Cysteine by Bound Ligand
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

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Materials

Basic Protocol: Reaction of Sulfhydryl Reagents with Substituted-Cysteine Mutanats

Materials

Ca²⁺- and Mg²⁺-free Dulbecco's phosphate-buffered saline (CMF-DPBS; unit 4.13)
CMF-DPBS /1 mM EDTA
Binding buffer (see recipe) with and without BSA, room temperature and ice cold
Radioligand (prepared in binding buffer; see appendix 3A for a typical radiolabeling procedure)
MTS reagent (Toronto Research Chemicals):
2-aminoethyl methanethiosulfonate hydrobromide (MTSEA; mol. wt. 236.2)
2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET; mol. wt. 278.2)
sodium 2-sulfanatoethyl methanethiosulfonate (MTSES; mol. wt. 233.2)
Unlabeled agonist and antagonist (prepared in binding buffer)

Glass fiber filters (934AH, particle retention 1.5 µm, Brandel)
Brandel cell harvester

Additional reagents and equipment for site-directed mutagenesis (units 4.10 & 4.11), restriction mapping (cpmb units 3.1-3.3 and appendix 1A in this manual), DNA sequencing (cpmb units 7.1-7.6 and appendix 1A in this manual), transient or stable expression of mutant proteins (units 4.5-4.7), radioligand saturation studies (units 7.5-7.7)

Support Protocol: Protection of Substituted Cysteine by Bound Ligand

Additional Materials (also see Basic Protocol)

96-well multiscreen plate containing GF/B filters (Millipore)

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Figures

Figure 4.15.1

Reaction of an MTS reagent with a Cys sulfhydryl at the water-accessible surface of a receptor.

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Figure 4.15.2

Schematic representation of the reaction of an MTS reagent with a Cys exposed in the binding-site crevice.The membrane is represented by the shaded rectangle, the binding-site crevice by the white area within the plane of the membrane, and ligand by the solid oval. SEX represents SCH₂CH₂X, where X is NH₃⁺, N(CH₃)₃⁺, or SO₃^–. SEX is covalently linked to the water-accessible Cys sulfhydryl. In the bound state (lower left panel), ligand is reversibly bound at the binding site within the binding-site crevice. In the unbound state (upper left), the binding site is unoccupied. After irreversible reaction with MTSEX (upper right), ligand can no longer bind (lower right). The Cys sulfhydryl facing lipid or the interior of the protein does not react significantly with MTSEX. MTSEX only reacts with a sulfhydryl in the binding-site crevice of ligand-free receptor. Thus, ligand binding retards the rate of reaction of receptor with MTSEX and protects subsequent ligand binding.

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Figure 4.15.3

A representative experiment showing the inhibition of ligand binding by reaction of MTSEA with a substituted Cys in the ₂ adrenoceptor. The data were fit by nonlinear regression using Prism (GraphPad) to a one-phase exponential decay function, Y = (span × e^–Kc) + plateau, where Y is the fraction of initial binding, c is the concentration of MTSEA and span + plateau = 1. The curve starts at (span + plateau) = 1, and decays to plateau at saturating MTSEA. In this sample fit, span = 0.81, K = 1382 M^–1, and plateau = 0.19. (r² = 0.97) since K = kt, where k is the second-order rate constant in M^–1 s^–1 and t is the time in sec (120 sec) of the reaction with the MTS reagents, k = K/t = 1382/120 = 11.5 M^–1s^–1.

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Literature Cited

Literature Cited
	Akabas, M.H., Stauffer, D.A., Xu, M., and Karlin, A. 1992. Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258:307-310.
	Chen, J.G., Sachpatzidis, A., and Rudnick, G. 1997. The third transmembrane domain of the serotonin transporter contains residues associated with susbstrate and cocaine binding. J. Biol. Chem. 272:28321-28327.
	Ferrer, J.V. and Javitch, J.A. 1998. Cocaine alters the accessibility of endogenous cysteines in putative extracellular and intracellular loops of the human dopamine transporter. Proc. Natl. Acad. Sci.U.S.A. 95:9238-9243.
	Fu, D., Ballesteros, J.A., Weinstein, H., Chen, J., and Javitch, J.A. 1996. Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice. Biochemistry 35:11278-11285.
	Javitch, J.A. 1998. Probing the structure of neurotransmitter transporters by the substituted-cysteine accessibility method. Methods Enzymol. 296:331-346.
	Javitch, J.A., Fu, D., and Chen, J. 1995a. Residues in the fifth membrane-spanning segment of the dopamine D2 receptor exposed in the binding-site crevice. Biochemistry 34:16433-16439.
	Javitch, J.A., Fu, D., Chen, J., and Karlin, A. 1995b. Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method. Neuron 14:825-831.
	Javitch, J.A., Fu, D., and Chen, J. 1996. Differentiating dopamine D2 ligands by their sensitivities to modification of the cysteine exposed in the binding-site crevice. Mol. Pharm. 49:692-698.
	Javitch, J.A., Fu, D., Liapakis, G., and Chen, J. 1997. Constitutive activation of the beta2 adrenergic receptor alters the orientation of its sixth membrane-spanning segment. J. Biol. Chem. 272:18546-18549.
	Javitch, J.A., Ballesteros, J.A., Weinstein, H., and Chen, J. 1998. A cluster of aromatic residues in the sixth membrane-spanning segment of the dopamine D2 receptor is accessible in the binding- site crevice. Biochemistry 37:998-1006.
	Jung, K., Jung, H., Wu, J., Prive, G.G., and Kaback, H.R. 1993. Use of site-directed fluorescence labeling to study proximity relationships in the lactose permease of Escherichia coli. Biochemistry 32:12273-12278.
	Karlin, A. and Akabas, M.H. 1998. Substituted-cysteine accessibility method. Methods Enzymol. 293:123-145.
	Rees, S., Coote, J., Stables, J., Goodson, S., Harris, S., and Lee, M.G. 1996. Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. BioTechniques 20:102-110.
	Roberts, D.D., Lewis, S.D., Ballou, D.P., Olson, S.T., and Shafer, J.A. 1986. Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry 25:5595-5601.
	Stauffer, D.A. and Karlin, A. 1994. Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. Biochemistry 33:6840-6849.
Key References
	Karlin and Akabas, 1998. See above.
This review covers the synthesis, chemistry, and reaction schemes of the MTS reagents as well as details of applying SCAM to ion channels.
	Javitch et al., 1995b. See above.
This was the first application of SCAM to a GPCR.
	Javitch, J.A. 1999. The substituted-cysteine accessibility method. In Structure/Function Analysis of G-Protein Coupled Receptors (J. Wess, ed.) pp. 21-42. John Wiley & Sons, New York.
This reviews the application of SCAM to dopamine D2 and ₂ adrenergic receptors.