The polypeptide gramicidin A is particularly appropriate for these studies of conformational stability, because it has a large surface to volume ratio. Its molecular structure and dynamics are highly sensitive to its environment. In lipid bilayers, gramicidin typically forms a single-stranded helical dimer (see Fig. 1, blue), whereas in organic solvents, it forms a variety of stable double-helical conformations (see Fig. 1, red) that vary in handedness and symmetry (i.e., parallel vs. antiparallel) (7). These conformers interconvert slowly enough in ethanol that four different conformations are well resolved by solution NMR spectroscopy (8). They convert much more slowly in dioxane or tetrahydrofuran, because the solvent cannot efficiently promote hydrogen bond exchange, and consequently, the conformers are trapped in a metastable state (7, 9, 10). In fact, they can be separated by TLC or normal-phase HPLC, and solution NMR spectroscopy can be performed on the individual conformers (10). The equilibrium between these conformational states varies, depending primarily on the dielectric constant of the solvent (unpublished results). Furthermore, when lipid bilayers containing gramicidin were prepared by first cosolubilizing peptide and lipid in various organic solvents followed by solvent removal and hydration, a solvent history dependenceThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.was observed (9,11,12 Solid-state NMR is well suited for characterizing proteins in anisotropic environments, and moreover, it can take advantage of uniaxially aligned samples (13-18). The gramicidin channel structure has been solved with solid-state NMR-derived orientational constraints from samples in fully hydrated lipid bilayers (13). Through isotope labeling of individual specific amino acid sites, orientation-dependent nuclear spin interactions have been monitored in aligned samples (see Fig. 1). Because of the many precise constraints observed for the gramicidin channel, its structure is one of the highest resolution characterizations of a membranebound polypeptide or protein structure. MATERIALS AND METHODSIsotopically labeled amino acids were purchased from Cambridge Isotope Laboratories (Cambridge, MA). Dimyristoyl phosphatidylcholine was purchased from Sigma. Both amino acids and lipids were used without further purification. Gramicidin A was synthesized using fluorenylmethoxycarbonyl blocking chemistry and a peptide synthesizer (Applied Biosystems; model 430A) (19). Peptides that were synthesized in <98% purity were purified by semipreparative HPLC as described (19). Oriented samples were prepared using a gramicidin/lipid molar ratio of 1:8. The peptide and lipid were cosolubilized in an organic solvent, and the sample was dried on thin glass plates. Approximately 20 plates were stacked in a segment of square glass tubing that was sealed following the addition of ...

A method, the metric method, is presented for determining the structure of polypeptides using solid state nuclear magnetic resonance (NMR) dipolar interactions. In analogy to the method of distance geometry used for protein structure determination from high resolution NMR spectroscopy, which is based on the general relationships that distances between points must satisfy, a method is developed here which makes use of the general relationships that angles between vectors must satisfy. With this method, analytical expressions are derived for the dihedral angles of the peptide backbone, and a way for minimizing the structural ambiguities associated with solid state NMR data is also presented. Calculations on a model polypeptide structure reveal the dipolar interactions of only the NH and NC1 bonds provide insufficient information for uniquely determining dihedral angles, even when constraints arising from long range interactions are employed, but these calculations yield a manageable number of solutions when the CαH interaction is also considered.

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High-resolution orientational constraints from solid-state NMR spectroscopy of uniformly aligned biological macromolecules provide a great structural analysis problem. Several approaches to this problem have been made in the past. Here a vector algebra method is developed that provides analytical solutions for the torsion angles and a concise and simple view of the structural possibilities. Numerical instabilities in this approach are easily predicted. Insight into how the structural ambiguities arise in the first place and how they can be reduced in number is demonstrated with this new approach.