A method for determining transmembrane helix association and orientation in detergent micelles using small angle x-ray scattering.
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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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.
Engelman
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D055592:Biophysical Phenomena; D001703:Biophysics; D002087:Butyrates; D003902:Detergents; D019281:Dimerization; D004563:Electrochemistry; D006021:Glycophorins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D008970:Molecular Weight; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012542:Scattering, Radiation; D012996:Solutions; D012997:Solvents; D014965:X-Rays
C093272:(N-dodecyl-N,N-dimethylammonio)butyric acid; D002087:Butyrates; D003902:Detergents; D006021:Glycophorins; D008565:Membrane Proteins; D008823:Micelles; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D012996:Solutions; D012997:Solvents