High-resolution polypeptide structure in a lamellar phase lipid environment from solid state NMR derived orientational constraints.

BACKGROUND

Solid-state nuclear magnetic resonance (NMR) spectroscopy provides novel structural constraints from uniformly aligned samples. These orientational constraints orient specific atomic sites with respect to the magnetic field direction and the unique molecular axis of alignment. Solid-state NMR is uniquely and ideally suited for providing such structural constraints on polypeptides and proteins in a lamellar phase lipid environment. Membrane protein structure represents a great challenge for structural biologists; a new approach for characterizing high resolution three-dimensional structure in such an environment is needed.

RESULTS

The optimal use of orientational constraints for defining three-dimensional structures is demonstrated with the elucidation of the gramicidin A channel structure at high resolution. Initial structures are refined against both the experimental constraints and the CHARMM energy using a novel simulated-annealing protocol to define torsion angle solutions with an error bar of approximately +/- 5 degrees.

CONCLUSIONS

This analysis results in the determination of a high-resolution, time averaged structure of gramicidin A obtained in a lipid bilayer environment above the gel-to-liquid crystalline phase transition temperature. It is demonstrated that solid-state NMR can be used to establish polypeptide, and potentially protein, structures in such an environment. Furthermore, this high-resolution structure is demonstrated to provide new insights into polypeptide function. For the gramicidin A channel the roles of the indole groups that facilitate ion transport and details of the cation solvation environment provided by the amide oxygens are characterized.