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GRAMICIDIN CHANNELS AND PORES B. A. Wallace Department of Chemistry and Center for Biophysics, Rensselaer Polytechnic Institutc, Troy, New York 12180 KEY WORDS:
membrane proteins, channels, ion transport, membranes.
CONTENTS PERSPECTIVES AND OVERVIEW .............. .. ............ ... .. .. .................... .... .............. ... .. ..........
127
CHEMICAL NATURE OF THE GRAMICIDINS . .............. .. ........... .... ... .... ... ............... ... .. .. ...... ..
128
MODEL NOMENCLATURE.................................................................................................
129
POLYMORPHISM OF GRAMICIDIN ...... ........ ............... .. ................ . . ...... .. ....... .... .................
133 133 137 139
Identification of Multiple Conformations in Solution . ............. .... ... ... .... ..... ... .. ....... ... One Predominant Conformation Is Present in Membranes............................ ............ Ion Binding Affects Gramicidin Conformations ... ......... ..... .. ... ........... .. ..... .. ............... PHYSICAL NATURE OF THE CONFORMERS ......... ................... .... ............ ..... .. .... ..................
The Pore Is a Double Helix ...................................................................................... The Channel Is a Helical Dimer................................................................................ fon Binding Sites in the Pore and in the Channel....................... ................................
141 1 41 144
147
RELATIONSHIP BETWEEN THE MOLECULAR SPECIES AND THE CONDUCTING FORMS.............
149
MEMBRANE INSERTION AND STABILITy ............................................................................
151
GENERAL IMPLICATIONS FOR ION CHANNELS ...
153
PERSPECTIVES AND OVERVIEW
Gramicidin is a linear polypeptide antibiotic that forms specific channels in membranes for the transport of monovalent cations. It is the most thoroughly characterized ion channel in terms of its electrical properties, the relationships between its chemical structure and conductance prop erties, and the details of its three-dimensional structure. It has been the basis for numerous theoretical studies modeling channel activity, ion inter actions, and dynamics. Although it was first isolated in 1940 (46), its 127 0883-9182/90/0610-0127$02.00
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WALLACE
A
Figure 1
B
Schematic diagrams of the gramicidin in the (a) channel (helical dimer) and
(b) pore (double helix) conformations.
chemical structure elucidated in 1964 (88), and the first crystals of it reported in 1949 (25, 45a), only in the past few years has great progress been made in understanding the relationship between structure and function for this "simple" ion channel. The availability of sensitive methods for measuring properties of individual channels; synthetic techniques for pre paring analogs; and developments in such physical techniques as nuclear magnetic resonance, fluorescence, and circular dichroism spectroscopies, and in X-ray diffraction have provided new insight into this molecule. Despite gramicidin's relatively simple chemistry, determining the nature of its three-dimensional structure has been neither straightforward nor facile. Because of its size and flexibility, this molecule adopts families of different types of conformations, depending on its environment, instead of a single conformation. The two major types of gramicidin con formations have been designated the "channel" and "pore" structures (Figure 1); these correspond to the forms primarily found in membranes and in organic solutions, respectively. A number of reviews have focused on the conductance properties of gramicidin (1, 33) and on gramicidin lipid interactions (22, 52). This review concentrates on recent insights gained into the structural features of the channel and pore forms of gramicidin and into structure-function relationships for this molecule. CHEMICAL NATURE OF THE GRAMICIDINS
Gramicidin denotes the group of hydrophobic linear polypeptides syn thesized by Bacillus brevis during sporulation (46). All members of the
GRAMICIDIN CHANNELS ANO PORES
129
group are composed of fifteen hydrophobic amino acids plus a carboxy terminal ethanolamine residue (a glycine without the carbonyl oxygen) and have their amino-termini blocked with a formyl group. Alternating amino acids along their chains are of different chiralities. They have the common sequence (88, 90):
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formyl-L-val-glY-L-ala-o-leu-L-ala-o-val-L-val-o-valL-trp-o-leu-L-xxx-D-leu-L-trp-D-Ieu-L-trp-ethanolamine, where xxx is tryptophan in gramicidin A, phenylalanine in gramicidin B (91), and tryosine in gramicidin C (89). Gramicidin D (Dubos) is the name given to the natural mixture of gramicidins A, B, and C, which are present in an approximate ratio of 80 : 5: 15 (122). The gramicidins have an additional heterogeneity in their first amino acid: in the natural mix, approximately 90% of all gramicidins have valine at this position, while about 10% have isoleucine (39). Most experiments to date have been done on this six-component mixture, and it is referred to as gramicidin in this review. Gramicidin K, which has a fatty acid covalently attached to the ethanolamine residue, can also be isolated with gramicidin D in varying amounts in different preparations (62). A strain of Bacillus brevis also synthesizes gramicidin S (Soviet), a cyclic decapeptide (38) that forms ion carriers. However, this molecule is not related to the linear gramicidins, either structurally or functionally. Hence, this review focuses solely on the linear gramicidins. Because the primary structure of gramicidin has neither charged nor hydrophilic side chains, and because both the amino- and carboxy-termini of the molecule are blocked (thereby preventing the formation of a zwitter ionic polypeptide at any pH), this molecule is almost insoluble in water ( < 10 mg/liter) but partitions strongly into the hydrophobic region of phospholipid membranes. It has little solubility in hydrocarbons, although it is highly soluble in a wide variety of alcohols, organic acids, and other organic solvents such as dimethylsulfoxide, acetone, dioxane, and tri fiuoroethanol. It can be solubilized in water in the presence oflysolecithin, gangliosides, sodium dodecyl sulfate, octyl glucoside, and other detergent molecules. MODEL NOMENCLATURE
Because of the somewhat unusual chemical structure of this molecule, most notably the presence of alternating L- and D-amino acids, and its known transport characteristics, the gramicidin channel could be modeled early on, even in the absence of extensive structural information. Those
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models proved especially insightful when compared with current infor mation on the actual structures. No standard nomenclature has emerged for these structures because gramicidin does not form one of the types of secondary structures commonly found in globular proteins. The following section correlates the various designations that have been applied to the gramicidin models and defines the terms used in the remainder of this article. In 1971, Urry proposed a helical structure for the gramicidin molecule, called a n4(L,D) helix (WI). This is a hybrid of a 4.416 and a 4.314 helix, where, in this nomenclature, the decimal (4.4) refers to the number of residues per turn, and the subscript (16) refers to the number of atoms per ring. In a structure with both L- and D-amino acids, alternating turns have a different number of atoms per ring, hence the hybrid designation. Ramachandran & Chandrasekaran (83) independently proposed a similar structural model based on empirical energy calculations. Urry et al later revised the model (103) to a noel, D) helix, which had a larger number of residues (6.3) per turn and a central hole ( � 4 A in diameter) that would be more appropriate for the size selectivity of the ions accommodated by the gramicidin channel than the 1.4-A diameter hole of a n4(L, D) helix. A dimer of polypeptide chains in the n 6(L, D) conformation would be 25-30 A in length, which approximates the size necessary to span a lipid bilayer. In this model, now known as the helical dimer (Figure la), two monomeric helices are associated with each other end-to-end. The helical dimer would form a cylindrical tube in which twelve parallel p-sheet-like hydrogen bonds between residues in adjacent turns produce the associations that stabilize each monomer. The association between monomers in a helical dimer could either be via the two amino-termini, the two carboxy-termini, or one amino- and one carboxy-terminus. The first two types of structures will result in symmetric dimers, whereas the latter will not have a center of symmetry and could potentially form a continuous spiral of multimers. In the amino-terminal-to-amino-terminal (otherwise known as the N-to-N, or head-to-head) dimer, the formyl groups reside at the bilayer center and the ethanolamine groups at the membrane surface. The opposite would be true for the C-to-C (tail-to-tail) dimer, whereas one amino-terminus and one carboxy-terminus would be at the membrane surface in the N-to-C dimcr modcl. The association between monomers in any of the n6(L, D) structures would be stabilized via six intermolecular hydrogen bonds. The original designation of this type of structure as a n helix was a bit of a misnomer: this nomenclature has traditionally referred to a 4.4 16 helix, with the typical helical orientations of the backbone hydrogen bonds (all amino groups pointing in the same direction, which results in a large
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GRAMICIDIN CHANNELS AND PORES
131
net dipole). The type of helix ascribed to gramicidin does not have that geometry; its amino groups point in alternating directions along the helix, which results in a net dipole near zero. In addition, the
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......
w Vl
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Each species had a distinct circular dichroism (CD) spectrum, which could provide information on the helical hands. Because the spectra of species 1 and 2 were similar, Veatch et al felt that they would have similar conformations (109). Species 1, 2, and 3 all had spectra with negative values in the wavelength range from 205 to 240 nm; the helical senses of these dimers thus were likely to be opposite from that of species 4, which had a nearly mirror-image spectrum to that of species 1 and 2. The spectrum obtained initially upon dissolution of crystals was that of species 3 (109). The spectrum of an equilibrium mixture (Figure 4) is an average of thc spectra of all the conformers present in solution, weighted by their relative abundances (109, 115). This net spectrum has a considerably lower magnitude than that of any of the individual spectra because the spectra of species 1, 2, and 4 nearly cancel each other out, producing an average magnitude close to zero, and a shape similar to that of species 3, even though species 3 is only a minor component. CD spectroscopy did not reveal the details of the conformations of each of the species, in part because the cp,!{I angles in (J helices are very different from those of rx helices, (J-sheets, or turns, and this makes comparisons with standard protein spectra ineffective. In addition, proteins normally have very low
10
-20
Figure 4
190
230 210 NAYEl.EN6TH Inll
250
270
Net circular dichroism spectra of the gramicidin in different environments illus in methanol (--) , and in trifluoroethanol (- - - -).
trating conformational flexibility in solution: in phospholipid vesicles (....),
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GRAMICIDIN CHANNELS AND PORES
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tryptophan contents so their aromatic signals in this region are negligible. This is not the case for gramicidin, in which tryptophan makes up 25% of its residues. Thus, transitions of the aromatic rings tend to obscure the peptide transitions, and dominate the gramicidin far ultraviolet CD spectra in the region around 220 nm. Gramicidin adopts similar mixtures of species in a variety of alcohols and ethyl acetate solutions (107). Although interconversion times are con siderably shorter in these solvents (II, 98, 107), multiple conformers were still distinguishable on the NMR time scale (16; 1. D. Callahan & B. A. Wallace, unpublished results). In contrast, thin layer chromatography detects only a single species in dimethylsulfoxide. Fluorescence (107) and high pressure liquid chroma tography (HPLC) (10) measurements show that it is mostly monomeric, and NMR studies have suggested (40) it is unordered structure that rapidly interconverts with a helical structure of a different sort than that found in the other solvents (perhaps with 4>, l/J angles like those in the helical dimer). The CD spectrum of gramicidin in trifluoroethanol (Figure 4) is entirely different from that obtained in other alcohols. Thin layer chromatography reveals only a single spot (S. Wormuth & B. A. Wallace, unpublished results) and osmometry measurements (47) suggest that gramicidin is a monomer. Killian et al (54) suggested that the CD spectrum in this solvent is more similar to that in membranes than that in any other organic solvent, but clearly, the structure in this environment must represent yet another conformation for the molecule. Thus, gramicidin adopts different conformations in different organic solvents, and even adopts a discrete mixture of different conformers in a single type of solvent. This may be because many of the conformers have similar stabilization energies, as they all have extensive (but differ ent) hydrogen-bonding networks, which give rise to specific secondary structures.
One Predominant Conformation Is Present in Membranes Gramicidin incotporates into phospholipid membranes to form trans membrane channels that conduct ions. The membrane-incorporated species has a distinct circular dichroism spectrum ( 119) that differs from that of the species found in !p ost organic solvents (Figure 4). It cannot be produced by any linear combmation of the solution spectra, and therefore does not represent a mixture of those species. From the CD spectrum alone, one cannot identify whether gramicidin adopts a single conformer or a mixture of conformers in membranes. However, similar net spectra obtained over a wide range of temperatures (119) strongly suggest a single species rather than an equilibrium mixture of species, whose relative
138
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proportions at different temperatures would be expected to change. NMR studies of 13C_ and 19F-Iabeled gramicidins incorporated into vesicles (120122) indicate the presence of one dominant conformation in membranes, although a small proportion (up to 10%) of another species could have gone undetected in those measurements, given the signal-to-noise levels. The far ultraviolet CD spectrum of the membrane-bound form of gramicidin has an exciton-like splitting pattern, which suggests tryptophan stacking in this environment (115). Exciton splitting can also be seen in the near ultraviolet region of the spectrum (1. D. Callahan & B. A. Wallace, submitted), resulting in a complicated spectrum that indicates intra molecular interactions. (No such excitonic splittings can be seen in the spectra of gramicidin in solution, another demonstration of the different types of conformations present in membranes and in organic solutions.) Fluorescence energy transfer (58) and anisotropy (93) measurements also indicate tryptophan stacking in the membrane-associated form, but not in the solution form. These results then suggest there are differences in both the backbone and side-chain conformations of gramicidin in. membranes and in solution. Simultaneous fluorescence and conductance studies of gramicidin in black lipid membranes (110) and phospholipid vesicles (111) have shown that the predominant conducting form in membranes is a dimer. Tn single channel conductance studies, where very low levels of polypeptide (gramicidin-to-lipid ratios of about 1: 10,000) are used, the openings and closing of the channels reveal the monomer-dimer equilibrium (Figure 7). However, under the relatively high polypeptide concentrations (ratios of between 1: 4 and 1: 400) used in most of the physical characterization measurements, essentially only the dimer form is present at all times (111). Gramicidin also forms complexes with detergent molecules such as lysolecithin (70) and sodium dodecylsulfate (SDS) (4). Freeze fracture electron microscopic studies indicated that the complexes form multi lamellar vesicles (53, 81), rather than the typical micellar complexes these detergents form with other proteins and polypeptides. Furthermore, low angle X-ray diffraction (53) and phosphorus (53) and deuterium (49) NMR studies demonstrated that the amphiphiles in these complexes adopt bilayer-like organizations (in the extended multilamellar structures). This unusual behavior is an example of the influence of gramicidin on the ordering of surrounding lipid and detergent molecules, a topic reviewed recently by Killian & de Kruijff (52). Because the detergents form bilayer like complexes rather than micelles, it was expected that the structure of gramicidin in this environment might be similar to that in phospholipid vesicles, whereas other small peptides often have very different structures in the matrices formed by bilayers and micelles. Indeed, the CD spectra
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�
GRAMICIDIN CHANNELS AND PORES
139
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of the lysolecithin and SDS complexes are more similar to the spectrum of gramicidin in vesicles than that of gramicidin in any of the organic solutions (4, 70). However, the spectra are not identical to the vesiele spectrum, and so in these environments, the conformation of gramicidin must differ somewhat from the channel structure. Gramicidin appears to adopt dimeric species in bilayers that are struc turally very different from the dimers found in solution.
Ion Binding Affects Gramicidin Conformations Gramicidin transports monovalent cations across phospholipid mem branes (45). Its differential selectivity for members of the alkali metal series relates to both the energy of hydration and size of these ions. The relative binding affinities of sodium, potassium, rubidium, cesium, and thallium for the ion channel have been estimated from single channel conductance measurements (31), channel profiles of analogs (71), NMR spectroscopy (44), equilibrium dialysis (108), and water permeability measurements (26). Thallium binding is one order of magnitude tighter than cesium, and up to two orders of magnitude tighter than sodium. Apparently this selectivity is not based on size exclusion, but rather is due to the higher energies required for dehydration. While trace flux measurements (82) show that at least two ions can occupy the channel simultaneously, dialysis measure ments (108) indicate that the association constant for the first ion is considerably higher than for the second, which suggests electrostatic repul sion between the two positively charged ions. Divalent cations block con ductance of monovalent cations (7), but are not themselves transported by the channel (7, 45). Recent studies demonstrated that partially dehydrated divalent cations can bind to the channel (42) with substantially higher binding cnthalpies than the monovalent ions. The strong interactions of divalent ions with the polypeptide may be responsible for their diminished mobility through the channel. Binding of anions to the channel form has not been demonstrated, although some evidence for anion permeability and anion influence on cation permeability exists (30, 31). The binding of monovalent cations to gramicidin in phospholipid ves icles has little or no effect on the net CD spectrum of the molecule (113). Therefore, the backbone fold of the channel form must not be significantly changed upon ion binding. Lysolecithin complexes of gramicidin also bind ions and have been used extensively to characterize the binding constants (41, 43) and determine the binding site locations (104, 106). These types of samples have size and fluidity characteristics that make them more amenable than phospholipid vesicles to the physical techniques employed in the binding studies. However, in contrast to the observations in vesicles, the CD spectrum of
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140
WALLACE
gramicidin in lysolecithin or in SDS shows a small change upon binding cations, suggesting a subtle change in the backbone conformation of the molecule (4, lO4), further evidence that the detergent-bound form of gramicidin may represent a species distinct from the channel. In organic solvents such as methanol, the CD spectra of gramicidin change dramatically upon binding monovalent cations (115). The spectra change in both magnitude and sign, suggesting a large alteration in the structure of the molecule. In contrast to the multiple species found for ion free gramicidin in solution, only a single species can be identified by NMR for gramicidin-cesium complexes (3; J. D. Callahan & B. A. Wallace, submitted). Gramicidin-CsC! crystals (117) have two crystallographically distinct dimers, but the rms deviation between all atoms in the two dimers is less than 0.1 A, and both dimers have essentially the same backbone folds with only slight differences in side-chain orientations. Consequently, these two dimers would not be readily detected as distinct species in solution. The CD results suggest a significant change in conformation between the ion-free and ion-bound forms. Comparisons of the crystal structures bear this out (65, 117). Normal mode analyses (75, 77) on crystals with and without ions identified large alterations in the molecular parameters upon ion binding. The binding of saturating concentrations of lithium ions causes similar but smallcr changes in the CD spectra, perhaps because less extensive changes are needed to accommodate the smaller lithium ions (56, 115). From crystal characteristics, we can discern infor mation about the relative effects of binding other types of ions on the gramicidin pore structure: potassium-containing crystals (Table 1) appear to be isomorphous with one type of cesium-containing crystals (grami cidin-CsSCN) (59), suggesting a similar structure; thallium-containing crystals (Table 1) are isomorphous with a different type of cesium-con taining crystals (gramicidin-CsCI-I) (56, 59), which.are believed to represent a form of the molecule with a smaller helical pitch. The CD spectrum of the pore alters upon binding a single cesium ion, and the ion-free form converts to the ion-bound form in a monotonic manner, implying a simple structural transition between the two conforma tions (115; J. D. Callahan & B. A. Wallace, submitted). These spectroscopic titrations have been used to calculate a cesium binding constant for the first tightest site (KI = 170 M- I ) and to estimate the binding constant for a second, weaker site (K2:::::: 20 M-1). The crystal structure of the gramicidin-CsCI complex confirms that two cations reside within the pore (117). The binding constant for lithium, measured in a similar manner, is at Icast one order of magnitude less tight than that for cesium (115). The binding constant for sodium (4 M-1) has been measured by 23Na-NMR (21). Binding constants for other monovalent cations to the pore form of
GRAMICIDIN CHANNELS AND PORES
141
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gramicidin have not yet been determined, a consequence of their lower solubilities and affinities for the gramicidin, which render such measure ments difficult. The available data, however, show the binding affinities of ions for the pore foIIow the same general trend as those for the channel. The different structural consequences of ion binding to the various conformations of gramicidin are a further demonstration of the con formational flexibility of this molecule. PHYSICAL NATURE OF THE CONFORMERS
Recent studie s employing diffraction, spectroscopic, and computational modeling techniques and conductance studies using chemically modified analogs have provided insight into the detailed structures of the pore and channel forms of gramicidin. Results from these widely diverse methods give very consistent views of the various conformers, which can now be compared with the models for the structure. Originally two alternate structural motifs were proposed for gramicidin: the helical dimer and the double helix. In retrospect, we can see that these very different structures (which formed the basis of the major controversy in the field) are both correct, depending on the environment in which gramicidin is found. The early models proposed for gramicidin correspond closely to thc gencral folding motifs found in the structures as we now know them. However, many of the features that the modeling studies did not anticipate also appear to be important for structural integrity and functional properties, and have been elucidated in recent structural studies. The Pore Is
a
Double Helix
The family of double helical structures formed by gramicidin in solution has been designated the "pore" conformation. The secondary structures of the four interconverting species present in ethanol solution in the absence of ions have been elucidated by two-dimensional NMR (16). Species 1 and 2 are left-handed parallel double helices, each with 5.6 residues per turn. They appear to differ from each other in the stagger between their chains but have the same helical sense and pitch. Species 2 has more ragged" ends, produced by a stagger of 3 residues. Species 4 is a right-handed paraIIel double helix, with the same p itch and stagger of the polypeptide chains as species I (and hence the reason for its mirror image CD spectrum). Species 3, the form obtained immediately upon solubilization of crystals (before they interconvert), is a left-handed anti parallel double helix with 5.6 resi dues per turn and a stagger of 3; it represents the most thermodynamically stable form in the crystal environ me nt. All are held together by 28 intermolecular hydrogen bonds (and "
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142
WALLACE
have no intramolecular hydrogen bonds). They are very similar to the models originally proposed by Veatch et aI, based on 'vibrational and electronic spectroscopy. The backbone fold of species 3 in solution is like that found in the crystal structure of ion-free gramicidin (65). However, the form present in the crystal structure represents only a minor component of all the species present in solution ( � 45%). Two-dimensional NMR studies of a cesium thiocyanate complex in methanol-chloroform suggest that, in this environment, the secondary structure is a right-handed antiparallel double helix, with a hydrogen bonding pattern like that in models with 7.2 residues per turn. The chains are offset by a stagger of 1.5 residues, which means that val-l does not form any hydrogen bonds within the dimer (3). This differs from the structure of the gramicidin-CsCI complex seen in crystals formed from methanol (117). In the crystal, the structure of the ion complex is a left handed antiparallel double helix with 6.4 residues per turn, and an align ment of polypeptide chains so that the ends are not ragged (stagger of 3). The 6.4 helix actually has the same hydrogen-bonding alignments as the 7.2 model, but differs in the superhelical twist of the polypeptide chains. Whether this subtlety could have been distinguished in the NMR measure ments is not clear. Among possible reasons for the other reported differ ences between the solution and solid-state structures are that different anions were present in the two environments [although this is unlikely given the isomorphic nature of the gramicidin-CsCI and gramicidin-CsSCN crystals (85)] or that different solvent systems were used (different ion bind ing constants and different NMR spectra are obtained in methanol and in methanol-chloroform) (1. D. Callahan & B. A. Wallace, submitted). Alter natively, like the ion-free crystals, the dimers in the ion-bound crystals could represent a less densely populated state than the predominant one seen in solution. The crystal structures of gramicidin provide additional details on its secondary structures and information on the tertiary structures and side chain conformations. Furthermore, the availability of crystals formed in both the presence and absence of ions permits examination of the effects of ions on the pore structure. Both the ion-bound and ion-free forms are left-handed antiparallel double helices, but they differ substantially in ¢, ljJ angles (Table 2), hydrogen-bonding patterns, helical pitches, and regularity of the backbones (Figure 5). The stagger of the chains are similar, and both give rise to structures with flat ends, but the form without ions is a long (31 A) thin pore with a central hole that is too narrow in some regions to accommodate ions (Figure Sa). Its polypeptide backbone has substantial irregularities or bulges along its length. Gramicidin crystallized in thc presence of cesium chloride is a shorter (26 A), fatter (�4.9 A diameter) structure in which cesiums are visible in the center of the pore (Figure 5b).
GRAMICIDIN CHANNELS AND PORES Table 2
Comparison of predicted and actual structures of the gramicidin pore
Residues/turn
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Models
Average
Annu.
Rev. Biophys. Biophys. Chem.
Copyright ©
1990 by Annual
1990. 19:127-57 Inc. All rights reserved
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GRAMICIDIN CHANNELS AND PORES B. A. Wallace Department of Chemistry and Center for Biophysics, Rensselaer Polytechnic Institutc, Troy, New York 12180 KEY WORDS:
membrane proteins, channels, ion transport, membranes.
CONTENTS PERSPECTIVES AND OVERVIEW .............. .. ............ ... .. .. .................... .... .............. ... .. ..........
127
CHEMICAL NATURE OF THE GRAMICIDINS . .............. .. ........... .... ... .... ... ............... ... .. .. ...... ..
128
MODEL NOMENCLATURE.................................................................................................
129
POLYMORPHISM OF GRAMICIDIN ...... ........ ............... .. ................ . . ...... .. ....... .... .................
133 133 137 139
Identification of Multiple Conformations in Solution . ............. .... ... ... .... ..... ... .. ....... ... One Predominant Conformation Is Present in Membranes............................ ............ Ion Binding Affects Gramicidin Conformations ... ......... ..... .. ... ........... .. ..... .. ............... PHYSICAL NATURE OF THE CONFORMERS ......... ................... .... ............ ..... .. .... ..................
The Pore Is a Double Helix ...................................................................................... The Channel Is a Helical Dimer................................................................................ fon Binding Sites in the Pore and in the Channel....................... ................................
141 1 41 144
147
RELATIONSHIP BETWEEN THE MOLECULAR SPECIES AND THE CONDUCTING FORMS.............
149
MEMBRANE INSERTION AND STABILITy ............................................................................
151
GENERAL IMPLICATIONS FOR ION CHANNELS ...
153
PERSPECTIVES AND OVERVIEW
Gramicidin is a linear polypeptide antibiotic that forms specific channels in membranes for the transport of monovalent cations. It is the most thoroughly characterized ion channel in terms of its electrical properties, the relationships between its chemical structure and conductance prop erties, and the details of its three-dimensional structure. It has been the basis for numerous theoretical studies modeling channel activity, ion inter actions, and dynamics. Although it was first isolated in 1940 (46), its 127 0883-9182/90/0610-0127$02.00
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A
Figure 1
B
Schematic diagrams of the gramicidin in the (a) channel (helical dimer) and
(b) pore (double helix) conformations.
chemical structure elucidated in 1964 (88), and the first crystals of it reported in 1949 (25, 45a), only in the past few years has great progress been made in understanding the relationship between structure and function for this "simple" ion channel. The availability of sensitive methods for measuring properties of individual channels; synthetic techniques for pre paring analogs; and developments in such physical techniques as nuclear magnetic resonance, fluorescence, and circular dichroism spectroscopies, and in X-ray diffraction have provided new insight into this molecule. Despite gramicidin's relatively simple chemistry, determining the nature of its three-dimensional structure has been neither straightforward nor facile. Because of its size and flexibility, this molecule adopts families of different types of conformations, depending on its environment, instead of a single conformation. The two major types of gramicidin con formations have been designated the "channel" and "pore" structures (Figure 1); these correspond to the forms primarily found in membranes and in organic solutions, respectively. A number of reviews have focused on the conductance properties of gramicidin (1, 33) and on gramicidin lipid interactions (22, 52). This review concentrates on recent insights gained into the structural features of the channel and pore forms of gramicidin and into structure-function relationships for this molecule. CHEMICAL NATURE OF THE GRAMICIDINS
Gramicidin denotes the group of hydrophobic linear polypeptides syn thesized by Bacillus brevis during sporulation (46). All members of the
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group are composed of fifteen hydrophobic amino acids plus a carboxy terminal ethanolamine residue (a glycine without the carbonyl oxygen) and have their amino-termini blocked with a formyl group. Alternating amino acids along their chains are of different chiralities. They have the common sequence (88, 90):
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formyl-L-val-glY-L-ala-o-leu-L-ala-o-val-L-val-o-valL-trp-o-leu-L-xxx-D-leu-L-trp-D-Ieu-L-trp-ethanolamine, where xxx is tryptophan in gramicidin A, phenylalanine in gramicidin B (91), and tryosine in gramicidin C (89). Gramicidin D (Dubos) is the name given to the natural mixture of gramicidins A, B, and C, which are present in an approximate ratio of 80 : 5: 15 (122). The gramicidins have an additional heterogeneity in their first amino acid: in the natural mix, approximately 90% of all gramicidins have valine at this position, while about 10% have isoleucine (39). Most experiments to date have been done on this six-component mixture, and it is referred to as gramicidin in this review. Gramicidin K, which has a fatty acid covalently attached to the ethanolamine residue, can also be isolated with gramicidin D in varying amounts in different preparations (62). A strain of Bacillus brevis also synthesizes gramicidin S (Soviet), a cyclic decapeptide (38) that forms ion carriers. However, this molecule is not related to the linear gramicidins, either structurally or functionally. Hence, this review focuses solely on the linear gramicidins. Because the primary structure of gramicidin has neither charged nor hydrophilic side chains, and because both the amino- and carboxy-termini of the molecule are blocked (thereby preventing the formation of a zwitter ionic polypeptide at any pH), this molecule is almost insoluble in water ( < 10 mg/liter) but partitions strongly into the hydrophobic region of phospholipid membranes. It has little solubility in hydrocarbons, although it is highly soluble in a wide variety of alcohols, organic acids, and other organic solvents such as dimethylsulfoxide, acetone, dioxane, and tri fiuoroethanol. It can be solubilized in water in the presence oflysolecithin, gangliosides, sodium dodecyl sulfate, octyl glucoside, and other detergent molecules. MODEL NOMENCLATURE
Because of the somewhat unusual chemical structure of this molecule, most notably the presence of alternating L- and D-amino acids, and its known transport characteristics, the gramicidin channel could be modeled early on, even in the absence of extensive structural information. Those
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models proved especially insightful when compared with current infor mation on the actual structures. No standard nomenclature has emerged for these structures because gramicidin does not form one of the types of secondary structures commonly found in globular proteins. The following section correlates the various designations that have been applied to the gramicidin models and defines the terms used in the remainder of this article. In 1971, Urry proposed a helical structure for the gramicidin molecule, called a n4(L,D) helix (WI). This is a hybrid of a 4.416 and a 4.314 helix, where, in this nomenclature, the decimal (4.4) refers to the number of residues per turn, and the subscript (16) refers to the number of atoms per ring. In a structure with both L- and D-amino acids, alternating turns have a different number of atoms per ring, hence the hybrid designation. Ramachandran & Chandrasekaran (83) independently proposed a similar structural model based on empirical energy calculations. Urry et al later revised the model (103) to a noel, D) helix, which had a larger number of residues (6.3) per turn and a central hole ( � 4 A in diameter) that would be more appropriate for the size selectivity of the ions accommodated by the gramicidin channel than the 1.4-A diameter hole of a n4(L, D) helix. A dimer of polypeptide chains in the n 6(L, D) conformation would be 25-30 A in length, which approximates the size necessary to span a lipid bilayer. In this model, now known as the helical dimer (Figure la), two monomeric helices are associated with each other end-to-end. The helical dimer would form a cylindrical tube in which twelve parallel p-sheet-like hydrogen bonds between residues in adjacent turns produce the associations that stabilize each monomer. The association between monomers in a helical dimer could either be via the two amino-termini, the two carboxy-termini, or one amino- and one carboxy-terminus. The first two types of structures will result in symmetric dimers, whereas the latter will not have a center of symmetry and could potentially form a continuous spiral of multimers. In the amino-terminal-to-amino-terminal (otherwise known as the N-to-N, or head-to-head) dimer, the formyl groups reside at the bilayer center and the ethanolamine groups at the membrane surface. The opposite would be true for the C-to-C (tail-to-tail) dimer, whereas one amino-terminus and one carboxy-terminus would be at the membrane surface in the N-to-C dimcr modcl. The association between monomers in any of the n6(L, D) structures would be stabilized via six intermolecular hydrogen bonds. The original designation of this type of structure as a n helix was a bit of a misnomer: this nomenclature has traditionally referred to a 4.4 16 helix, with the typical helical orientations of the backbone hydrogen bonds (all amino groups pointing in the same direction, which results in a large
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net dipole). The type of helix ascribed to gramicidin does not have that geometry; its amino groups point in alternating directions along the helix, which results in a net dipole near zero. In addition, the
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m Vl
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w Vl
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Each species had a distinct circular dichroism (CD) spectrum, which could provide information on the helical hands. Because the spectra of species 1 and 2 were similar, Veatch et al felt that they would have similar conformations (109). Species 1, 2, and 3 all had spectra with negative values in the wavelength range from 205 to 240 nm; the helical senses of these dimers thus were likely to be opposite from that of species 4, which had a nearly mirror-image spectrum to that of species 1 and 2. The spectrum obtained initially upon dissolution of crystals was that of species 3 (109). The spectrum of an equilibrium mixture (Figure 4) is an average of thc spectra of all the conformers present in solution, weighted by their relative abundances (109, 115). This net spectrum has a considerably lower magnitude than that of any of the individual spectra because the spectra of species 1, 2, and 4 nearly cancel each other out, producing an average magnitude close to zero, and a shape similar to that of species 3, even though species 3 is only a minor component. CD spectroscopy did not reveal the details of the conformations of each of the species, in part because the cp,!{I angles in (J helices are very different from those of rx helices, (J-sheets, or turns, and this makes comparisons with standard protein spectra ineffective. In addition, proteins normally have very low
10
-20
Figure 4
190
230 210 NAYEl.EN6TH Inll
250
270
Net circular dichroism spectra of the gramicidin in different environments illus in methanol (--) , and in trifluoroethanol (- - - -).
trating conformational flexibility in solution: in phospholipid vesicles (....),
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tryptophan contents so their aromatic signals in this region are negligible. This is not the case for gramicidin, in which tryptophan makes up 25% of its residues. Thus, transitions of the aromatic rings tend to obscure the peptide transitions, and dominate the gramicidin far ultraviolet CD spectra in the region around 220 nm. Gramicidin adopts similar mixtures of species in a variety of alcohols and ethyl acetate solutions (107). Although interconversion times are con siderably shorter in these solvents (II, 98, 107), multiple conformers were still distinguishable on the NMR time scale (16; 1. D. Callahan & B. A. Wallace, unpublished results). In contrast, thin layer chromatography detects only a single species in dimethylsulfoxide. Fluorescence (107) and high pressure liquid chroma tography (HPLC) (10) measurements show that it is mostly monomeric, and NMR studies have suggested (40) it is unordered structure that rapidly interconverts with a helical structure of a different sort than that found in the other solvents (perhaps with 4>, l/J angles like those in the helical dimer). The CD spectrum of gramicidin in trifluoroethanol (Figure 4) is entirely different from that obtained in other alcohols. Thin layer chromatography reveals only a single spot (S. Wormuth & B. A. Wallace, unpublished results) and osmometry measurements (47) suggest that gramicidin is a monomer. Killian et al (54) suggested that the CD spectrum in this solvent is more similar to that in membranes than that in any other organic solvent, but clearly, the structure in this environment must represent yet another conformation for the molecule. Thus, gramicidin adopts different conformations in different organic solvents, and even adopts a discrete mixture of different conformers in a single type of solvent. This may be because many of the conformers have similar stabilization energies, as they all have extensive (but differ ent) hydrogen-bonding networks, which give rise to specific secondary structures.
One Predominant Conformation Is Present in Membranes Gramicidin incotporates into phospholipid membranes to form trans membrane channels that conduct ions. The membrane-incorporated species has a distinct circular dichroism spectrum ( 119) that differs from that of the species found in !p ost organic solvents (Figure 4). It cannot be produced by any linear combmation of the solution spectra, and therefore does not represent a mixture of those species. From the CD spectrum alone, one cannot identify whether gramicidin adopts a single conformer or a mixture of conformers in membranes. However, similar net spectra obtained over a wide range of temperatures (119) strongly suggest a single species rather than an equilibrium mixture of species, whose relative
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proportions at different temperatures would be expected to change. NMR studies of 13C_ and 19F-Iabeled gramicidins incorporated into vesicles (120122) indicate the presence of one dominant conformation in membranes, although a small proportion (up to 10%) of another species could have gone undetected in those measurements, given the signal-to-noise levels. The far ultraviolet CD spectrum of the membrane-bound form of gramicidin has an exciton-like splitting pattern, which suggests tryptophan stacking in this environment (115). Exciton splitting can also be seen in the near ultraviolet region of the spectrum (1. D. Callahan & B. A. Wallace, submitted), resulting in a complicated spectrum that indicates intra molecular interactions. (No such excitonic splittings can be seen in the spectra of gramicidin in solution, another demonstration of the different types of conformations present in membranes and in organic solutions.) Fluorescence energy transfer (58) and anisotropy (93) measurements also indicate tryptophan stacking in the membrane-associated form, but not in the solution form. These results then suggest there are differences in both the backbone and side-chain conformations of gramicidin in. membranes and in solution. Simultaneous fluorescence and conductance studies of gramicidin in black lipid membranes (110) and phospholipid vesicles (111) have shown that the predominant conducting form in membranes is a dimer. Tn single channel conductance studies, where very low levels of polypeptide (gramicidin-to-lipid ratios of about 1: 10,000) are used, the openings and closing of the channels reveal the monomer-dimer equilibrium (Figure 7). However, under the relatively high polypeptide concentrations (ratios of between 1: 4 and 1: 400) used in most of the physical characterization measurements, essentially only the dimer form is present at all times (111). Gramicidin also forms complexes with detergent molecules such as lysolecithin (70) and sodium dodecylsulfate (SDS) (4). Freeze fracture electron microscopic studies indicated that the complexes form multi lamellar vesicles (53, 81), rather than the typical micellar complexes these detergents form with other proteins and polypeptides. Furthermore, low angle X-ray diffraction (53) and phosphorus (53) and deuterium (49) NMR studies demonstrated that the amphiphiles in these complexes adopt bilayer-like organizations (in the extended multilamellar structures). This unusual behavior is an example of the influence of gramicidin on the ordering of surrounding lipid and detergent molecules, a topic reviewed recently by Killian & de Kruijff (52). Because the detergents form bilayer like complexes rather than micelles, it was expected that the structure of gramicidin in this environment might be similar to that in phospholipid vesicles, whereas other small peptides often have very different structures in the matrices formed by bilayers and micelles. Indeed, the CD spectra
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of the lysolecithin and SDS complexes are more similar to the spectrum of gramicidin in vesicles than that of gramicidin in any of the organic solutions (4, 70). However, the spectra are not identical to the vesiele spectrum, and so in these environments, the conformation of gramicidin must differ somewhat from the channel structure. Gramicidin appears to adopt dimeric species in bilayers that are struc turally very different from the dimers found in solution.
Ion Binding Affects Gramicidin Conformations Gramicidin transports monovalent cations across phospholipid mem branes (45). Its differential selectivity for members of the alkali metal series relates to both the energy of hydration and size of these ions. The relative binding affinities of sodium, potassium, rubidium, cesium, and thallium for the ion channel have been estimated from single channel conductance measurements (31), channel profiles of analogs (71), NMR spectroscopy (44), equilibrium dialysis (108), and water permeability measurements (26). Thallium binding is one order of magnitude tighter than cesium, and up to two orders of magnitude tighter than sodium. Apparently this selectivity is not based on size exclusion, but rather is due to the higher energies required for dehydration. While trace flux measurements (82) show that at least two ions can occupy the channel simultaneously, dialysis measure ments (108) indicate that the association constant for the first ion is considerably higher than for the second, which suggests electrostatic repul sion between the two positively charged ions. Divalent cations block con ductance of monovalent cations (7), but are not themselves transported by the channel (7, 45). Recent studies demonstrated that partially dehydrated divalent cations can bind to the channel (42) with substantially higher binding cnthalpies than the monovalent ions. The strong interactions of divalent ions with the polypeptide may be responsible for their diminished mobility through the channel. Binding of anions to the channel form has not been demonstrated, although some evidence for anion permeability and anion influence on cation permeability exists (30, 31). The binding of monovalent cations to gramicidin in phospholipid ves icles has little or no effect on the net CD spectrum of the molecule (113). Therefore, the backbone fold of the channel form must not be significantly changed upon ion binding. Lysolecithin complexes of gramicidin also bind ions and have been used extensively to characterize the binding constants (41, 43) and determine the binding site locations (104, 106). These types of samples have size and fluidity characteristics that make them more amenable than phospholipid vesicles to the physical techniques employed in the binding studies. However, in contrast to the observations in vesicles, the CD spectrum of
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gramicidin in lysolecithin or in SDS shows a small change upon binding cations, suggesting a subtle change in the backbone conformation of the molecule (4, lO4), further evidence that the detergent-bound form of gramicidin may represent a species distinct from the channel. In organic solvents such as methanol, the CD spectra of gramicidin change dramatically upon binding monovalent cations (115). The spectra change in both magnitude and sign, suggesting a large alteration in the structure of the molecule. In contrast to the multiple species found for ion free gramicidin in solution, only a single species can be identified by NMR for gramicidin-cesium complexes (3; J. D. Callahan & B. A. Wallace, submitted). Gramicidin-CsC! crystals (117) have two crystallographically distinct dimers, but the rms deviation between all atoms in the two dimers is less than 0.1 A, and both dimers have essentially the same backbone folds with only slight differences in side-chain orientations. Consequently, these two dimers would not be readily detected as distinct species in solution. The CD results suggest a significant change in conformation between the ion-free and ion-bound forms. Comparisons of the crystal structures bear this out (65, 117). Normal mode analyses (75, 77) on crystals with and without ions identified large alterations in the molecular parameters upon ion binding. The binding of saturating concentrations of lithium ions causes similar but smallcr changes in the CD spectra, perhaps because less extensive changes are needed to accommodate the smaller lithium ions (56, 115). From crystal characteristics, we can discern infor mation about the relative effects of binding other types of ions on the gramicidin pore structure: potassium-containing crystals (Table 1) appear to be isomorphous with one type of cesium-containing crystals (grami cidin-CsSCN) (59), suggesting a similar structure; thallium-containing crystals (Table 1) are isomorphous with a different type of cesium-con taining crystals (gramicidin-CsCI-I) (56, 59), which.are believed to represent a form of the molecule with a smaller helical pitch. The CD spectrum of the pore alters upon binding a single cesium ion, and the ion-free form converts to the ion-bound form in a monotonic manner, implying a simple structural transition between the two conforma tions (115; J. D. Callahan & B. A. Wallace, submitted). These spectroscopic titrations have been used to calculate a cesium binding constant for the first tightest site (KI = 170 M- I ) and to estimate the binding constant for a second, weaker site (K2:::::: 20 M-1). The crystal structure of the gramicidin-CsCI complex confirms that two cations reside within the pore (117). The binding constant for lithium, measured in a similar manner, is at Icast one order of magnitude less tight than that for cesium (115). The binding constant for sodium (4 M-1) has been measured by 23Na-NMR (21). Binding constants for other monovalent cations to the pore form of
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gramicidin have not yet been determined, a consequence of their lower solubilities and affinities for the gramicidin, which render such measure ments difficult. The available data, however, show the binding affinities of ions for the pore foIIow the same general trend as those for the channel. The different structural consequences of ion binding to the various conformations of gramicidin are a further demonstration of the con formational flexibility of this molecule. PHYSICAL NATURE OF THE CONFORMERS
Recent studie s employing diffraction, spectroscopic, and computational modeling techniques and conductance studies using chemically modified analogs have provided insight into the detailed structures of the pore and channel forms of gramicidin. Results from these widely diverse methods give very consistent views of the various conformers, which can now be compared with the models for the structure. Originally two alternate structural motifs were proposed for gramicidin: the helical dimer and the double helix. In retrospect, we can see that these very different structures (which formed the basis of the major controversy in the field) are both correct, depending on the environment in which gramicidin is found. The early models proposed for gramicidin correspond closely to thc gencral folding motifs found in the structures as we now know them. However, many of the features that the modeling studies did not anticipate also appear to be important for structural integrity and functional properties, and have been elucidated in recent structural studies. The Pore Is
a
Double Helix
The family of double helical structures formed by gramicidin in solution has been designated the "pore" conformation. The secondary structures of the four interconverting species present in ethanol solution in the absence of ions have been elucidated by two-dimensional NMR (16). Species 1 and 2 are left-handed parallel double helices, each with 5.6 residues per turn. They appear to differ from each other in the stagger between their chains but have the same helical sense and pitch. Species 2 has more ragged" ends, produced by a stagger of 3 residues. Species 4 is a right-handed paraIIel double helix, with the same p itch and stagger of the polypeptide chains as species I (and hence the reason for its mirror image CD spectrum). Species 3, the form obtained immediately upon solubilization of crystals (before they interconvert), is a left-handed anti parallel double helix with 5.6 resi dues per turn and a stagger of 3; it represents the most thermodynamically stable form in the crystal environ me nt. All are held together by 28 intermolecular hydrogen bonds (and "
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have no intramolecular hydrogen bonds). They are very similar to the models originally proposed by Veatch et aI, based on 'vibrational and electronic spectroscopy. The backbone fold of species 3 in solution is like that found in the crystal structure of ion-free gramicidin (65). However, the form present in the crystal structure represents only a minor component of all the species present in solution ( � 45%). Two-dimensional NMR studies of a cesium thiocyanate complex in methanol-chloroform suggest that, in this environment, the secondary structure is a right-handed antiparallel double helix, with a hydrogen bonding pattern like that in models with 7.2 residues per turn. The chains are offset by a stagger of 1.5 residues, which means that val-l does not form any hydrogen bonds within the dimer (3). This differs from the structure of the gramicidin-CsCI complex seen in crystals formed from methanol (117). In the crystal, the structure of the ion complex is a left handed antiparallel double helix with 6.4 residues per turn, and an align ment of polypeptide chains so that the ends are not ragged (stagger of 3). The 6.4 helix actually has the same hydrogen-bonding alignments as the 7.2 model, but differs in the superhelical twist of the polypeptide chains. Whether this subtlety could have been distinguished in the NMR measure ments is not clear. Among possible reasons for the other reported differ ences between the solution and solid-state structures are that different anions were present in the two environments [although this is unlikely given the isomorphic nature of the gramicidin-CsCI and gramicidin-CsSCN crystals (85)] or that different solvent systems were used (different ion bind ing constants and different NMR spectra are obtained in methanol and in methanol-chloroform) (1. D. Callahan & B. A. Wallace, submitted). Alter natively, like the ion-free crystals, the dimers in the ion-bound crystals could represent a less densely populated state than the predominant one seen in solution. The crystal structures of gramicidin provide additional details on its secondary structures and information on the tertiary structures and side chain conformations. Furthermore, the availability of crystals formed in both the presence and absence of ions permits examination of the effects of ions on the pore structure. Both the ion-bound and ion-free forms are left-handed antiparallel double helices, but they differ substantially in ¢, ljJ angles (Table 2), hydrogen-bonding patterns, helical pitches, and regularity of the backbones (Figure 5). The stagger of the chains are similar, and both give rise to structures with flat ends, but the form without ions is a long (31 A) thin pore with a central hole that is too narrow in some regions to accommodate ions (Figure Sa). Its polypeptide backbone has substantial irregularities or bulges along its length. Gramicidin crystallized in thc presence of cesium chloride is a shorter (26 A), fatter (�4.9 A diameter) structure in which cesiums are visible in the center of the pore (Figure 5b).
GRAMICIDIN CHANNELS AND PORES Table 2
Comparison of predicted and actual structures of the gramicidin pore
Residues/turn
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Models
Average
