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. 1999 Aug;77(2):1064–1073. doi: 10.1016/S0006-3495(99)76956-0

A method for determining transmembrane helix association and orientation in detergent micelles using small angle x-ray scattering.

Z Bu 1, D M Engelman 1
PMCID: PMC1300396  PMID: 10423450

Abstract

Solution small angle x-ray scattering can be used to study the association of transmembrane proteins solubilized in detergent micelles. We have used the alpha-helical transmembrane domain of the human erythrocyte glycophorin A (GpA) fused to the carboxyl terminus of monomeric staphylococcal nuclease (SN/GpA) as a model system for study. By matching the average electron density of the detergent micelles to that of the buffer solution, the micelle contribution to the small angle scattering vanishes, and the molecular weight and the radius of gyration of the proteins can be determined. SN/GpA has been found to dimerize in a zwitterionic detergent micelle, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), whose average electron density naturally matches the electron density of an aqueous buffer. The dimerization occurs through the transmembrane domains of GpA. With the aid of the nuclease domain scattering, the orientation of the helices within a dimer can be determined to be parallel by radius of gyration analysis. The association constant of a mutant (G83I) that weakens the GpA dimerization has been determined to be 24 microM in the DDMAB environment. The experimental methods established here could be used to apply solution small angle x-ray scattering to studying the association and interactions of other membrane proteins.

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Selected References

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  1. Adair B. D., Engelman D. M. Glycophorin A helical transmembrane domains dimerize in phospholipid bilayers: a resonance energy transfer study. Biochemistry. 1994 May 10;33(18):5539–5544. doi: 10.1021/bi00184a024. [DOI] [PubMed] [Google Scholar]
  2. Bormann B. J., Knowles W. J., Marchesi V. T. Synthetic peptides mimic the assembly of transmembrane glycoproteins. J Biol Chem. 1989 Mar 5;264(7):4033–4037. [PubMed] [Google Scholar]
  3. Brenner C., Jan G., Chevalier Y., Wróblewski H. Evaluation of the efficacy of zwitterionic dodecyl carboxybetaine surfactants for the extraction and the separation of mycoplasma membrane protein antigens. Anal Biochem. 1995 Jan 20;224(2):515–523. doi: 10.1006/abio.1995.1081. [DOI] [PubMed] [Google Scholar]
  4. Flanagan J. M., Kataoka M., Fujisawa T., Engelman D. M. Mutations can cause large changes in the conformation of a denatured protein. Biochemistry. 1993 Oct 5;32(39):10359–10370. doi: 10.1021/bi00090a011. [DOI] [PubMed] [Google Scholar]
  5. Jan G., Brenner C., Wróblewski H. Purification of Mycoplasma gallisepticum membrane proteins p52, p67 (pMGA), and p77 by high-performance liquid chromatography. Protein Expr Purif. 1996 Mar;7(2):160–166. doi: 10.1006/prep.1996.0023. [DOI] [PubMed] [Google Scholar]
  6. Jeanteur D., Pattus F., Timmins P. A. Membrane-bound form of the pore-forming domain of colicin A. A neutron scattering study. J Mol Biol. 1994 Jan 21;235(3):898–907. doi: 10.1006/jmbi.1994.1047. [DOI] [PubMed] [Google Scholar]
  7. Langosch D., Brosig B., Kolmar H., Fritz H. J. Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator. J Mol Biol. 1996 Nov 8;263(4):525–530. doi: 10.1006/jmbi.1996.0595. [DOI] [PubMed] [Google Scholar]
  8. Leeds J. A., Beckwith J. Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interactions in vivo. J Mol Biol. 1998 Jul 31;280(5):799–810. doi: 10.1006/jmbi.1998.1893. [DOI] [PubMed] [Google Scholar]
  9. Lemmon M. A., Flanagan J. M., Hunt J. F., Adair B. D., Bormann B. J., Dempsey C. E., Engelman D. M. Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices. J Biol Chem. 1992 Apr 15;267(11):7683–7689. [PubMed] [Google Scholar]
  10. Lemmon M. A., Flanagan J. M., Treutlein H. R., Zhang J., Engelman D. M. Sequence specificity in the dimerization of transmembrane alpha-helices. Biochemistry. 1992 Dec 29;31(51):12719–12725. doi: 10.1021/bi00166a002. [DOI] [PubMed] [Google Scholar]
  11. MacKenzie K. R., Prestegard J. H., Engelman D. M. A transmembrane helix dimer: structure and implications. Science. 1997 Apr 4;276(5309):131–133. doi: 10.1126/science.276.5309.131. [DOI] [PubMed] [Google Scholar]
  12. MacKenzie K. R., Prestegard J. H., Engelman D. M. Leucine side-chain rotamers in a glycophorin A transmembrane peptide as revealed by three-bond carbon-carbon couplings and 13C chemical shifts. J Biomol NMR. 1996 May;7(3):256–260. doi: 10.1007/BF00202043. [DOI] [PubMed] [Google Scholar]
  13. Pachence J. M., Edelman I. S., Schoenborn B. P. Low-angle neutron scattering analysis of Na/K-ATPase in detergent solution. J Biol Chem. 1987 Jan 15;262(2):702–709. [PubMed] [Google Scholar]
  14. Perkins S. J., Weiss H. Low-resolution structural studies of mitochondrial ubiquinol:cytochrome c reductase in detergent solutions by neutron scattering. J Mol Biol. 1983 Aug 25;168(4):847–866. doi: 10.1016/s0022-2836(83)80078-3. [DOI] [PubMed] [Google Scholar]
  15. Pessen H., Kumosinski T. F., Timasheff S. N. Small-angle x-ray scattering. Methods Enzymol. 1973;27:151–209. doi: 10.1016/s0076-6879(73)27011-8. [DOI] [PubMed] [Google Scholar]
  16. Russ W. P., Engelman D. M. TOXCAT: a measure of transmembrane helix association in a biological membrane. Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):863–868. doi: 10.1073/pnas.96.3.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Trewhella J. Insights into biomolecular function from small-angle scattering. Curr Opin Struct Biol. 1997 Oct;7(5):702–708. doi: 10.1016/s0959-440x(97)80081-4. [DOI] [PubMed] [Google Scholar]

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