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 t he at-helical transmembrane domain of the human erythrocyte glycophorin A ( GpA) fused to the carboxyl terminus of monomeric staphylococcal nuclease (S N/GpA) as a model system for study. By matching the average electron densit y of the detergent micelles to that of the buffer solution, the micelle con tribution to the small angle scattering vanishes, and the molecular weight and the radius of gyration of the proteins can be determined. SN/GpA has be en found to dimerize in a zwitterionic detergent micelle, N-dodecyl-N,N-(di methylammonio)butyrate (DDMAB), whose average electron density naturally ma tches the electron density of an aqueous buffer. The dimerization occurs th rough the transmembrane domains of GpA. With the aid of the nuclease domain scattering, the orientation of the helices within a dimer can be determine d to be parallel by radius of gyration analysis. The association constant o f a mutant (G83I) that weakens the GpA dimerization has been determined to be 24 mu M in the DDMAB environment. The experimental methods established h ere could be used to apply solution small angle x-ray scattering to studyin g the association and interactions of other membrane proteins.