Conformational trapping in a membrane environment: a regulatory mechanism for protein activity?

Abstract

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equilibrium between these conformational states varies, depending primarily on the dielectric constant of the solvent (unpublished results).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 ...

The effect of temperature and lipid on the conformational transition of gramicidin A in lipid vesicles

“…This model implies the existence of an intermediate between the double-stranded helix and single-stranded helical dimmer. In general, the present mechanism is more consistent with the flip-flop model, 26,27 since the ''zipper'' model involves several sequential steps and implies the existence of a series of intermediates. 24,25,29 The conformational change of gramicidin A in lipid vesicles is an extremely slow process compared to the usual water-soluble proteins, and this is reflected in the low values of the two rate constants between 10 À1 and 10 À3 per minute.…”

“…In the second model, referred to as the ''flip-flop'' mechanism, the intertwined chains twist to dissociate, and the peptide planes reorient and flip to produce a structure with the opposite helical handedness. 26,27 However, the detailed mechanism underlying this structural conversion under the effect of temperature and lipid has not been fully studied.…”

Validation of the single-stranded channel conformation of gramicidin A by solid-state NMR

“…Moreover, the structure in the membrane-mimetic SDS micelles is definitively single-stranded, and the solid-state NMR structure described here in lamellar phase lipids is also definitively single-stranded. The only structurally characterized doublestranded conformer of gramicidin A in a lipid bilayer was shown to be in a kinetically trapped state that, on heating at 68°C for 3 days, was converted to the single-stranded channel state (16).…”

Water: Foldase activity in catalyzing polypeptide conformational rearrangements

“…This realization has led to the recent finding of kinetically trapped polypeptide conformational states in lipid bilayer environments (9,10) and to the hypothesis of protic solvent catalysis of hydrogen bond exchange (5). To investigate the role of water as a catalyst for hydrogen bond exchange and as a ''foldase'' in nonpolar environments, we have studied solvent-dependent conformational transitions of gramicidin A.…”