Location and orientation relative to the micelle surface for glucagon in mixed micelles with dodecylphosphocholine EPR and NMR studies

The spin labels, 5-doxylstearate, 12-doxylstearate, 16-doxylstearate and 1-oxyl-2,2,6,6-tetramethyl-4-dodecylphospiperidine, have been incorporated into dodecylphospocholine micelles and mixed dodecylphosphocholine/ glucagon micelles. The EPR spectral parameters for the different spin labels and the ¹H- and ¹³C-NMR relaxation rates for nuclei of the detergent molecules indicated that inclusion of up to one spin label molecule per micelle had little influence on the spatial organization of the micelles. Furthermore, the location and environment of the spin labels in the dodecylphosphocholine micelles were not noticeably affected by the addition of glucagon and the ¹H-NMR spectra observed for glucagon in mixed spin label/deuterated dodecylphosphocholine/glucagon micelles showed that the different spin labels had essentially no effect on the conformation of glucagon. Approximate spatial locations within the micelle for the nitroxide moieties of the different spin labels were determined from the NMR relaxation rates observed for different nuclei of dodecylphosphocholine. On this basis, the line broadening of individually assigned glucagon ¹H-NMR lines by the different spin labels was used to determine the approximate orientation of the polypeptide chain with respect to the micelle surface. Overall, the data indicate that the glucagon backbone runs roughly parallel to the micelle surface, with the depth of immersion adjusted so that polar and apolar side chains can be oriented towards the surface or interior of the micelle, respectively.