MACROMOLECULAR STRUCTURAL ELUCIDATION WITH SOLID-STATE NMR-DERIVED ORIENTATIONAL CONSTRAINTS

The complete structure determination of a polypeptide in a lipid bilay er environment is demonstrated built solely upon orientational constra ints derived from solid-state NMR observations. Such constraints are o btained from isotopically labeled samples uniformly aligned with respe ct to the B-0 field. Each observation constrains the molecular frame w ith respect to B-0 and the bilayer normal, which are arranged to be pa rallel. These constraints are not only very precise (a few tenths of a degree), but also very accurate. This is clearly demonstrated as the backbone structure is assembled sequentially and the i to i+6 hydrogen bonds in this structure of the gramicidin channel are shown on averag e to be within 0.5 Angstrom of ideal geometry. Similarly, the side cha ins are assembled independently and in a radial direction from the bac kbone. The lack of considerable atomic overlap between side chains als o demonstrates the accuracy of the constraints. Through this complete structure, solid-state NMR is demonstrated as an approach for determin ing three-dimensional macromolecular structure.