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

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

1 Columbia 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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Reaction of Sulfhydryl Reagents with Substituted‐Cysteine Mutanats
  • Support Protocol 1: Protection of Substituted Cysteine by Bound Ligand
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: 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 recipebinding 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 1.NaN in this manual), DNA sequencing (CPMB UNITS 7.1 7.6 and 1.NaN 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 1: Protection of Substituted Cysteine by Bound Ligand

  • 96‐well multiscreen plate containing GF/B filters (Millipore)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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 β2 adrenergic receptors.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library