Structure of Gramicidin A

STRUCTURE OF GRAMICIDIN A B. A. WALLACE Department ofBiochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 ABSTRACT Gramicidin A, a hydrophobic linear polypeptide, forms channels in phospholipid membranes that are specific for monovalent cations. Nuclear Magnetic Resonance (NMR) spectroscopy provided the first direct physical evidence that the channel conformation in membranes is an amino terminal-to-amino terminal helical dimer, and circular dichroism (CD) spectroscopy has shown the sensitivity of its conformation to different environments and the structural consequences of ion binding. The three-dimensional structure of a gramicidin/cesium complex has been determined by x-ray diffraction of single crystals using single wavelength anomalous scattering for phasing. The left-handed double helix in this crystal form corresponds to one of the intermediates in the process of folding and insertion into membranes. Co-crystals of gramicidin and lipid that appear to have gramicidin in their membrane channel conformation have also been formed and are presently under investigation. Hence, we have used a combination of spectroscopic and diffraction techniques to examine the conformation and functionally-related structural features of gramicidin A. INTRODUCTION cidin in alcohol and dioxane solutions, Veatch et al. (1974) Gramicidin A is a hydrophobic linear polypeptide anti- proposed an alternate family of models (Fig. 1 C, D), the biotic consisting of 15 amino acids with alternating L- and parallel and antiparallel intertwined double helices. None D-configurations. It has the sequence (Sarges and Witkop, of these studies, however, examined the structure of the 1965): molecule in phospholipid bilayers. While the molecular folds of the helical dimer and HCO-L-val-gly-L-ala-D-leu-L-ala-D-val-L-val-D-val-L-trp-D-leu- double-helical models are completely different, they have L-trp-D-leu-L-trp-D-leu-L-trp-NHCH2CH2OH. many similar physical characteristics, making distinguish- ing between these structures difficult. In the double helix, In membranes, gramicidin A forms channels that are the adjacent hydrogen bonds are intermolecular, while in specific for monovalent cations (Hladky and Haydon, the helical dimer, most are intramolecular. However, 1972); it is, to date, the physiologically best-characterized because the pitch of the double helix is twice that of the ion channel (Finkelstein and Andersen, 1981). Further- helical dimer, the chain separation per turn along the helix more, it is an excellent model system for conformational axis and the hydrogen bonding patterns are similar. Fur- studies of membrane proteins. Fluorescence and conduc- thermore, the lengths (-30 A), and outer (-15 A) and tance measurements in black lipid membranes and phos- inner channel diameters (-4 -5 A) of the two folding pholipid vesicles have shown that the active conducting motifs are nearly indistinguishable. One striking difference form of the molecule is a dimer (Veatch et al., 1975; between the two types of models is the location of their Veatch and Stryer, 1977). The structure of this dimer has amino- and carboxyl-termini relative to the bilayer surface. been the subject of investigation in a number of laborato- Model A has the carboxyl-termini exposed at the surface ries. and the amino-termini buried deep in the membrane. In In an early study, based, in part, on CD and NMR contrast, model B has the carboxyl-termini buried and the studies of the molecule in organic solvents, Urry first amino-termini exposed. Models C and D have all their proposed (Urry, 1971; Urry et al., 1971) a 7r(L,D) helix amino- and carboxyl-termini at the surface. conformation for the channel in which two helical monom- Hence, the gramicidin structure in membranes could be ers were associated in the membrane via their amino- identified by a method that differentially probed locations termini (Fig. 1 A). Conductance studies using chemically normal to the bilayer surface. "3C- and '9F-NMR studies of modified gramicidins in black lipid films formed from gramicidin in phospholipid vesicles were able to make such glycerol monooleate and decane (Szabo and Urry, 1979; distinctions (Weinstein et al., 1979; Weinstein et al., Bamberg et al., 1977) supported this model as well as, to 1980). Using the membrane-impermeable probes Tm++ some extent, a similar helical dimer with the carboxyl- and Mn+ +, which are located in the aqueous space termini associated instead (Bradley et al., 1978) (Fig. 1 B). surrounding the bilayer, it was possible to show that the Based primarily on IR and CD measurements of grami- carboxyl-termini were exposed at the membrane surface BIOPHYS. J. © Biophysical Society * 0006-3495/86/01/295/12 $1.00 295 Volume 49 January 1986 295-306 Type II Vesicles. For the binding and insertion studies, gramicidin was added to preformed sonicated DMPC vesicles as previously described (Wallace, 1984). IN For both types of vesicle preparations, control vesicles were prepared in the same manner without gramicidin present. All type I and II vesicles (both with and without gramicidin) were <500 A in diameter, as shown by negative stain electron microscopy. The differential light scattering and absorption flattening effects of these particles are small and will not significantly distort the resulting spectra (Mao and Wallace, 1984). Titrations A B C D Stock solutions ofgramicidin (16.5 mg/ml in methanol) were diluted with either 6 M LiCl or 110 mM CsCl plus the appropriate amount of FIGURE 1 Schematic diagrams of models for the gramicidin A dimer: methanol to a constant gramicidin concentration (1.5 mg/ml). (a) amino-to-amino helical dimer; (b) carboxyl-to-carboxyl helical dimer; (c) antiparallel double helix; and (d) parallel double helix. Crystallizations Gramicidin/cesium crystals were prepared in a manner similar to that and that the amino-termini were inaccessible to the surface previously described (Kimball and Wallace, 1984), except that the form probes, which is consistent with model A. However, the II crystals used for data collection were prepared by addition of 190 ,l of negative finding concerning the amino-termini was not CsCl solution (100 mM in methanol) and 60 Ml of methanol to 50,ul of conclusive because it could have been a result of, for gramicidin solution (150 mg/ml in methanol). example, steric hindrance to accessibility. To clarify this issue, another paramagnetic probe, located on the distal CD spectroscopy end of a phospholipid molecule, was used to determine the Spectra were collected on either a Cary 60 spectropolarimeter with a location of sites in the membrane interior. This probe had 6001 CD attachment or an Aviv (Lakewood, NJ) 60DS spectropolar- the opposite effect of the surface probes: the amino-termini imeter. Both instruments had a variable position detector. Spectra were of gramicidin, but not the carboxyl-termini, were obtained with the photomultiplier located directly adjacent to the sample affected, cell, resulting in a 900 acceptance angle, to reduce scattering effects. thus showing that the amino terminal ends of the polypep- Measurements were made at 25°C using 0.01 to 0.1 cm pathlength cells. tide are indeed buried in the lipid bilayer, while the At least three scans of two independent samples were collected and carboxyl termini are surface exposed, consistent only with averaged for each specimen. model A. This same sort of approach has been taken more recently by Urry et al. ( 1 983a) to locate ion binding sites of Diffraction gramicidin in detergent complexes, and to confirm this Preliminary characterizations of the crystals were done using Ni-filtered assignment of the orientation. Cu Ka radiation from a Philips x-ray generator operating at 40 kv, 30 ma. It is of interest to reconcile with these results the data 150 precession photographs were taken with a crystal-to-film distance of based on studies in organic solvents that suggested the 100 mm. The high-resolution (1.8 A) data set was collected on a single (0.16 x 0.16 x 0.1 mm) crystal at room temperature (16-190C) using alternate family of double helical folding motifs. That is, Ni-filtered Cu Ka radiation on a Picker FACS-1 diffractometer. Friedel does the molecule adopt the same conformation in isotrop- pairs were collected successively in blocks of 20 reflections. Details ofdata ic, hydrophilic organic solvents and in anisotropic, hydro- collection and processing will be described elsewhere (Wallace and phobic lipid bilayers? Also, what are the detailed struc- Hendrickson, manuscript in preparation). tures of the molecule in these two environments? RESULTS AND DISCUSSION MATERIALS AND METHODS To compare the structures of gramicidin in two different Materials environments, it was important to do parallel experiments Gramicidin (ICN Nutritional Biochemicals, Cleveland, OH), a mixture using a single physical technique. Circular dichroism spec- of 80% gramicidin A, 6% gramicidin B, and 14% gramicidin C, was troscopy permits direct comparison between gramicidin in crystallized from absolute ethanol and used without further purification. small unilamellar phospholipid vesicles and in solution. In this paper, this mixture will be refered to as gramicidin A. Gramicidin Veatch et al. (1974) had shown that several different concentrations were determined using the molar extinction coefficient interconvertible species are present in alcohol solutions and (e = 22,000) at 282 nm. Lipid concentrations were determined by phosphate assay (Fiske and Subbarow, 1925). All

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