Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance

J. Mol. Biol. (1992) 226, 1101-1109

Structure of an Isolated Gramicidin A Double Helical Species by High-resolution Nuclear Magnetic Resonance S. M. Pascal and T. A. Cross Institute

of Molecular Biophysics and Department Florida State University, Tallahassee, FL,

(Received

11 November

1991; accepted 8 April

of Chemistry U.S.A. 1992)

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and twodimensional nuclear Overhauser effect spectra at 509 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/ simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 57 residues per turn. Unique values for helical dimensions are also specified. Keywords: gramicidin;

n.m.r.; distance geometry;

simulated annealing; circular dichroism

switching the bacterium from vegetative growth to sporulation has been shown (Sarkar et al., 1977; There are many reasons why the determination of Fisher t Blumenthal, 1982). These two physiostructure for relatively small polypeptides is both logical roles undoubtedly require different concomplex and controversial. First, there are few formations since the former utilizes the formation of intramolecular hydrogen bonds and, because of the a channel conformation in a lipid environment and relatively small size of the peptide, these electrothe latter involves binding to the d subunit of RNA static interactions are highly solvent-accessible. polymerase in a partial aqueous environment. Consequently, they are easily disrupted in aqueous Although there is no crystal structure for the solution or other polar solvents. Second, the use of channel conformation, a wide variety of spectroorganic solvents in the studies raises the question of scopic data now documents that the structure in a non-physiological conditions. Third, small peptides lipid environment is a formyl end to formyl end are typically not compact enough to possess dimer, forming a single-stranded helix that spans a substantial tertiary structure. Hence, the longlipid bilayer. Recently, the helix sense has been range structure in such a molecule is very difficult to shown to be right-handed and the backbone torsion describe. angles for the first few residues have been deterEach of these issues is critical in a discussion of mined experimentally (Nicholson & Cross, 1989; the structure of gramicidin A. This molecule is a Teng et al., 1991). While the folding motif for the pentadecapeptide of alternating L and D amino channel conformation is well accepted, the structure acids produced by the bacterium, Bacillus brevis. of gramicidin bound to the polymerase is not. The sequence, including a blocking group on each Not only does gramicidin adopt multiple conend, is CHO-Val,-Gly-Ala,-Leun-Ala,-Val,-Val,formations in viva, but a wide variety of unrefined Vain-Trp,-Leuo-Trp,-Leuu-Trp,-Leun-Trp,structures have been published for in vitro environNHCH,CH,OH. It is one of the few polypeptides ments. Dimethyl sulfoxide and trifluoro ethanol with a known and well-characterized dual physioinduce substantially unfolded monomers (Roux et logical role. The lytic role of gramicidin as a monoal., 1990; Urry et aZ., 1972), while many organic valent cation selective channel has been extensively solvents (e.g. methanol, ethanol and dioxane) induce studied (Katz Q DeMain, 1977; Anderson, 1984). a heterogeneous mixture of several stable dimers. More recently, a regulatory role of gramicidin in Four different intertwined double-helical conforma1101

1. Introduction

0022-2836/92/16110149

$08.00/O

0

1992 Academic

Press

Limited

1102

S. M. Pascal

tions were proposed (Veatch et al., 1974), based upon infrared (i.r.f) spectroscopy, circular dichroism (c.d.) and one-dimensional solution nuclear magnetic resonance (n.m.r.) studies. The i.r. amide I band was interpreted in terms of proposed hydrogen bonding patterns, and cd. was used to determine the helix sense. These species interconvert rapidly in many organic solvents, but more slowly in dioxane. One of these species has been isolated and characterized in dioxane by two-dimensional n.m.r. techniques (Arseniev et al., 1984). A similar backbone conformation has since been obtained from a diffraction study performed on an uncomplexed crystalline preparation from a solution of benzene/ ethanol (Langs, 1988). This structure is a lefthanded, antiparallel, double-stranded helical dimer with 56 residues per turn. Three more species have been studied as part of a mixture of conformations in ethanol through the use of a shortened analog in dioxane (Arseniev et al., 1986). A second unrefined crystal structure has been determined with cesiumcomplexed gramicidin crystallized from methanol (Wallace & Ravikumar, 1988). This structure is also a left-handed, antiparallel, double-stranded helical dimer, but with 6.4 residues per turn, and a hydrogen bonding pattern identical to that for models with 7.2 residue per turn. The conformation isolated here exhibits a cd. spectrum similar to one of the species isolated by thin layer chromatography (Veatch et al., 1974) and indirectly characterized by Arseniev et al. (1986). Therefore, we will refer to this species as “species 4”, after these papers. Unlike cytoplasmic proteins, for which hydrophobic and van der Waals’ interactions are primarily responsible for stabilizing protein conformation, membrane-bound protein and polypeptide stability may be much more dependent on electrostatic interactions, especially hydrogen bonds. Consequently, solvents that can compete for the intramolecular hydrogen bonds destabilize the conformation, or catalyze the interconversion between discreet conformations. This behavior is well known for gramicidin in ethanol or methanol solvent systems (Veatch et al., 1974). In dioxane, several different conformations of gramicidin can be trapped, but the addition of water allows interconversion. For this hydrophobic peptide, studies in aqueous solution would be very difficult due to low solubility, and not necessarily appropriate because this is a non-physiological environment for this molecule. While a lipid environment can be chosen for the study of the channel state, one of the primary motivations for this study is to understand t Abbreviations used: Lr., infrared; c.d., circular dichroism; n.m.r., nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; h.p.l.c., high pressure liquid chromatography; WV., ultraviolet; t.l.c., thin-layer chromatography; HOHAHA, two-dimensional homonuclear Hartman Hahn spectroscopy; DQF-COSY, double-quantum filtered 2-dimensional correlated spectroscopy; NOESY, 2-dimensional nuclear Overhauser effect spectroscopy; p.p.m., parts per million; r.m.s.d., root-mean-square deviation.

and T. A. Cross

the solvent-dependent conformations of this peptide. Fundamentally, this is of interest for the determination of protein structure. Clearly, the axiom that the primary sequence dictates a unique global conformation is not always correct; one must also consider the environment of the polypeptide. Secondly, the presumed hydrophobic binding site on the RNA polymerase is not presently characterized. Thus, it is not clear at this time whether this environment would be better mimicked by aqueous or organic solution. A third reason to study gramicidin in organic solvents stems from the known solvent history dependence for the channel conformation (LoGrasso et al., 1988). When gramicidin and lipid are mixed in organic solvents, then dried and hydrated, the resultant gramicidin conformation is dependent on the organic solvent used, and hence: presumably, on the gramicidin conformation in the organic solvent. Consequently, one of the motivations of these studies is to understand which conformations can be interconverted to the channel state readily and which cannot, thereby gaining some clues as to how this peptide is refolded into the channel state. The study of gramicidin in an organic solvent has the typical problem for polypeptide structure determination of few tertiary structural constraints. In fact, almost all peptides of this size with welldefined structures either have proline residues or disulfide linkages confining their structure (e.g. see Dyson et al., 1988). Here, there are no such constraints. In fact, there are no identifiable intramolecular medium or long-range nuclear Overhauser enhancement values (NOE) and yet the molecule has a very well-defined conformation in the preparation described here.

2. Materials (a) Preparative

and Methods isolation

of

species

4

Pure valine gramicidin A was obtained by solid-phase peptide synthesis as described elsewhere (Fields et aE., 1988). Vortexing and heating to 60°C for 10 min was required to dissolve 200 mg of gramicidin A in 2 ml of a 100 : 1 h.p.1.c. grade dioxane/water solution (solvents were purchased from Aldrich). After a cooling period, 500+1 portions were eluted isocratically from a Beckman Ultra-sphere SI normal phase h.p.1.c. column at a flow rate of @5 ml/min (Fig. 1). The mobile phase was also a 100 : 1 dioxane/water solution. U.V. absorption at 320 nm was monitored as a function of retention time. Fractions collected every 20 s were analyzed via cd. spectra (Fig. 2) recorded on a J-5OOC spectropolarimeter (Jasco Inc., Easton MD). The fractions, which eluted at approximately 26 to 29 min (Fig. l), were the only fractions to show a positive ellipticity in the peptide backbone region near 230 nm, indicating a conformation distinct from the rest of the fractions. This conformation is referred to here as species 4, after Veatch et al. (1974), who performed a similar separation by preparative t.1.c. These fractions were pooled, dried in a speed-vat as soon aa possible to avoid interconversion of species, redissolved in 1,4-dioxane-ds (99%, Cambridge Isotopes), and transferred to a 5 mm n.m.r. tube. The last 2 steps were carried out, in a nitrogen-filled glove bag. The tube was then sealed to prevent absorption of oxygen and water.

Structure of an Isolated Gramicidin

1103

A Species

(b)

I

(a) 4

I

I

30

40 Minutes

Figure 1. h.p.1.c. chromatogram of gramicidin A. The peptide was eluted isocratically from a Beckman Ultra-sphere SI normal phase column with 100: 1 dioxene/water mobile phase, at a flow rate of 0.5 ml/min, and detected by U.V. absorption at 320 nm. c.d. spectra of fractions from region (a) and region (b) are shown in Fig. 2. L3U

L4U

nm

(b) Two-dimensional

n.m.r. spectra

n.m.r. spectra were recorded, non-spinning, on a 500 MHz Varian VXRBOO spectrometer, at a sample temperature of 30°C. All spectra were recorded in phasesensitive mode using the hyper-complex method (States et al., 1982). Recycle times were set to 2.0 s. HOHAHA (Braunschweiler & Ernst, 1983) spectra used an MLEV-17 mixing scheme (Bax & Davis, 1985) with the transmitter power set to give a 90” proton pulse of approximately 20 /.Is. The transmitter frequency was set at the center of the residual dioxane proton resonance. Most spectra were collected with 4096 points (2048 real) in t,, and 512 increments of t, (1024 spectra), except the DQFCOSY (Pisntini et al., 1982; Shaka & Freeman, 1983) and the 300 ms NOESY (Jeener et al., 1979; Kumar et al., 1980) used for assignment purposes (2048 points). The center of the residual dioxane resonance was referenced to 3.53 p.p.m. (c) Data analysis Spectra were processed on a Sun Spare station 1, using Varian VNMR sys4 software version 2.2. NOESY spectra were filtered by a Gaussian function. All others were filtered by sine-bells shifted 90” in fi and 60” in fi. All spectra were then zero filled to (4096 x 4096). Baseline correction, via a spline fit for predefined baseline regions, was performed in thefi dimension of all NOESY spectra used quantitatively. Distance geometry and simulated annealing were performed on a Silicon Graphics Personal Iris 4D/25TG, using DSPACE version 4.0 (Hare & Assoc.) and X-plor version 1.5 (Axe1 T. Brunger) aa described in Results.

Figure 2. c.d. spectra of h.p.1.c. fractions from Fig. 1 region (a) (top), and region (b) (bottom). The fractions with positive elliptic&y in this region (a) are referred to here as species 4, after Veatch et al. (1974). These fractions were pooled and analyzed by n.m.r. techniques.

3. Results (a) Sample Fractions collected from the leading peak of the h.p.1.c. chromatogram were pooled to form the n.m.r. sample. These fractions are clearly distinct from the other fractions, as shown by c.d. (Fig. 2). The conformational purity of the sample is clearly shown by the absence of an additional network of peaks in Figure 3. A few additional isolated weak peaks appear in the spectra, consistent with a small fraction of other conformations resulting from overlap of the h.p.1.c. peaks. (b) Spin assignments Near-complete proton resonance assignments (Table 1) were accomplished by standard procedures. Only the formyl proton, ethanolamine OH and the Leu” and Leu14 methyl groups were not assigned, due to spectral overlap. Individual spin systems were assigned via double quantum-filtered COSY (Fig. 3(a)) and 70 millisecond mixing time HOHAHA spectra. Other values of mixing time

1104

S. M. Pascal and T. A. Cross

n3= Via

f: EE16 ::

2 8-O d ci

.

A5 OB

18G2:‘:

2 ci d

8.0 0

-i,

3”

5-O

4-o

G2o .V8

6

9 WI3

6-O

o

*

*WI5

6.0

5-O

wI (p.p.m.1

WI (p.p.m.1

(a)

(b)

Figure 3. NHaH

region of (a) DQF-COSY spectrum and (b) 300 ms mixing time NOESY spectrum of species 4. Both spectra were recorded at 30°C and 500 MHz. Intraresidue cross-peaks are identified. The NOESY peaks for Wll and Et16 are below the plotted contour level. Backbone NOES are summarized in Fig. 4.

were not needed, at 70 milliseconds. proton NOES, at (Fig. 3(b)), were assignments.

since very few peaks were missing Sequential alpha proton to amide a mixing time of 306 milliseconds used to make sequence-specific

(c) Experimental

restraints (NOEs)

Next, a series of NOESY experiments were acquired with mixing times of 100, 150,266 and 250 milliseconds. A total of 188 useful NOES (160 involving side-chain resonances) were unambiguously identified. Intensities of NOE crosspeaks (I,,,) were determined by volume integration using VNMR sys4 software version 2.2, after subtraction of representative background volumes from nearby regions. All detected NOES were posi-

tive, indicating a correlation time >466 picoseconds. Buildup rates were linear, and distances were determined from the equation I,,, = kfr6. The constant k was determined for the 206millisecond NOESY spectrum by measurement of the Gly’ geminal aH-aH cross-peak, for which the assumed proton-proton distance is 1.76 A (1 A = 61 nm). For NOES involving tryptophan rings, k was determined from the indole-H to 8H cross-peak (2.53 A). The other 266millisecond NOES were then sorted into five categories: strong (2.0 to 2.2 A), medium + (2.0 to 2.5 A), medium - (2.0 to 3.0 A) weak + (2.5 to 4.0 A) and weak - (3.0 to 50 A). A very clear backbone NOE pattern emerged (Fig. 4). Medium-sized aN(i,i-2) NOES were found for nearly all even (i), while aN(i,i+2) NOES were found for all odd (i). There were no aN(i,i- 1) NOES detected. Almost all aN(i,i+ 1) NOES were strong.

Table 1 ‘H n.m.r. chemical ship assignments for gramicidin Residue V&l’ Gly’ Ala3 Leu4 Ala’ Va16 Val’ Val’ Trp’

NH 7566

8199 7.330

8471 8259 9094

Trp”

8548 8477 8621 9131 8558

I&U’2

9.126

Trp’3

8.876 8.809 8840 7.714

Led0

Lell-

Tl-p’S Et16 Chemical

shifts

Ha 4673

4153,

HB

HY

1.989

0.969

HS

A species 4 in dioxane

HEI

H&3

Hl2

W3

H82

9567

7.859

7223

6858

6.987

6.672

9443

7362

7.153

6.781

6946

6895

9386

7.657

7.175

6887

6.987

6.887

9.482

7.57 1

7.257

7-044

7.082

3845

5516 4408 5381

4818 5841 4084 5806 4664 5954 4780 5-733 4761 4504 4088

are expressed

1.188 1.726 1.316 2.397 2057 1.743 3117, 3016 1.574 3-083, 3079 1.755 3278,3106

1500 3109, 3042 3165 at 3O”C,

relative

0910

1.338

1.235, 1.014,

1.141 0.849

0733,@013 7.164

1.093

0199

1.394 1.352

to dioxane

at 353

p.p.m.

Structure

of an Isolated

‘aN(i,i-2)

‘aN(iit2)

aN(di+l)

aN(i.i)

l

NN(l;itl)

--I

Figure

3

4

5

6

7 8

9

IO II

12 13 14 15 16

of backbone NOES for species: NOE; () denotes a medium NOE. Intensities for aN( 12,12), aN( 12,lO) and aN( 14,12) were estimated due to spectral overlap. aN( 11,ll) was not observed, but atoms must be less than 31 A apart; (*) denotes intermolecular NOES (except for NN( 1,2)).

(m)

4.

2

denotes

Summary

a strong

(d) State of aggregation; monomer-speci$c

hydrogen

bonds;

assignments

Gramicidin A has been shown to form dimers in dioxane (Veatch & Blout, 1974; Sychev et al., 1980). Only one set of resonances was observed, but this could indicate either a monomer or a symmetric dimer. For a monomer conformation, the backbone NOE pattern (Fig. 4) is not consistent with any of the standard secondary structures for all L-amino acid proteins. The presence of n-amino acids alters the intensities, but not the existence or non-existence of these NOES. Assuming the presence of only a monomer, all NOE-derived distance constraints were entered into a simulated annealing routine (Nilges et al., 1988) using X-plor version 1.5 (Axe1 T. i+

i-l

r-l

i+r

;+#+I

i

Gramicidin

1105

A Species

Brunger) on a Silicon Graphics Personal Iris 4D/ 25TG. This routine models NOES as square-well restraints with force constants that increase as the run progresses. Resultant conformations were of high energy and non-convergent. NOE constraints could not be simultaneously satisfied. Computer modeling was used to study the possibility of a dimer conformation. An “ideal” p chain composed of only L-amino acids would have torsion angles & = - 120”, I,&= + 120”. These values would be reversed if the chain was composed of only n-amino acids: &,= + 120”, tin= - 120”. Alternating these values for an LD peptide would result in a “/I circle” structure, which resembles a fl chain except that it is curved and meets itself after a number of residues determined by the particular torsion angles (6.6 residues for the ideal values). All of the side-chains are outside of the LD circle, in contrast to an L-amino acid j? chain structure in which the side-chains alternate on either side of the chain. Figure 5 shows the hydrogen bonding pattern for a portion of a stacked pair of j? circles: a parallel b sheet pattern of hydrogen bonding between the circles, consisting of 12-membered rings. Throughout this discussion, i represents a n-amino acid (even i). If these circles had identical sequences, but were offset with respect to each other by x residues, then a pattern of hydrogen bonding, and therefore a predicted pattern of NOES, could be described as a function of x. Only the pattern predicted with x = 2 approximates the observed backbone NOES (Fig. 4). For this offset, the predominant intermolecular NOES are cr’N(i,i - 2) and aN’(i + 1 ,i + 3), where prime denotes top circle. Therefore, for species 4, we have assumed intermolecular hydrogen bonds of (NH(i) to CO’(i+l) and (NH’(i+3) to CO(i)). Each hydrogen bond was converted into two distance constraints: d,, = 1.9 A, d,, = 2.9 ii (Baker & Hubbard, 1984; Mitchell & Price, 1990). ;+#+I

i+x

i+x+1

;+I

Figure 5. Stereo view of a portion of 2 /l circles. Parallel B-sheet hydrogen bonding pattern is shown on the left stereo projection; approximate inter-chain NHaH dist,ances are shown on the right. Residue i is a D amino acid, and therefore even-numbered in the case of gramicidin A. The variable x represents the residue offset between the 2 chains. The observed backbone NOES require z = 2, thus specifying the hydrogen-bonding pattern.

1106

S. M. Pascal and T. A. Cross

Figure 6. Backbone superposition (stereo view) of 6 best distance geometry/simulated annealing structures calculated by DSPACE (see Results for protocol). Note the lack of fraying at the helical ends. Helix dimensions are summarized in Table 3. On the

of this model, backbone NOES were as intra- or intermolecular as noted in

basis

defined Figure

4.

With these backbone NOES and hydrogen bonds entered as distance constraints, the simulated annealing routine yielded regular right-handed intertwined parallel helices. These helices were much lower in overall energy than the monomer structures, and much better able to satisfy the NOE constraints. These preliminary structures were used as a basis for assigning each side-chain NOE as intra- or intermolecular. Generally, NOES from residue (i to i), (i to i + / - 1) are intramolecular; (i to i + / - 3,4. . . ) are intermolecular; (i to i + / - 2) NOES could be either intra- or intermolecular. Because of the exact coincidence of the chemical shifts, full symmetry was assumed. Therefore, each NOESY cross-peak was interpreted as two symmetric distance constraints. Higher levels of aggregation would likely break the chemical shift degeneracy of the separate molecules. (e) Structure Next, all NOES and hydrogen-bond distance constraints were entered into a distance geometry/ simulated annealing routine using DSPACE version 4.0. From a random embed, each residue was first subjected to 10, 50 and 100 cycles of conjugate gradient minimization, ignoring all distance constraints to other residues. This procedure was repeated, but for 2, 3, 4, 5, 6, 10 and 14-residue segments, then finally for the entire molecule. Next, 11 simulated annealing iterations with 106 cycles each were performed, with shake enabled and full

symmetry required between molecules. In the first iteration, Brownian motion was imparted to the atoms (heating), and damping was used when the penalty function rose above 60. The next ten iterations were also damped when the penalty function rose to above 60 for the first four, and above 30 for the last six iterations (cooling). Chiral centers were

fixed

and

100 cycles

of conjugate

gradient

minimization were performed. From 18 runs, ten right-handed double helices were generated (Table 2). The six best structures are shown in Figure 6 (backbone only). The average variability of the backbone atoms (r.m.s.d. from the Table 2 Analysis of DSPACE-derived right-handed helical structures No.

violationst

r.m.s.

1 0 0 1 3 3 4 2 0 1 Averages: 1.5

right-handed

Minimized 0

average

violati0ns.J

Penalty5

0.135 0132 0116 0.130 0130 0182 0175 0.136 0.114 0.133

143 13.2 lo-6 13.1 13.6 30.6 236 141 11.1 13.6

0138

158

0099

8.0

helices of 6 best

t Number of NOE violations $ r.rn.s. NOE violation. $ DSPACE penalty function.

greater

than

0.5 A.

Structure

of an Isolated

Gramicidin

Table 3 of the six structures

Analysis

Length?

Width$

27.2 27.3 27.6 26.6 27.5 26.3 Average 27.1 Values 27.5

No. residues/ turn

2.65 264 2.59 2.74 263 2.65 values

shown

575 575 575 575 575 575

structure

after 567

peptide preparations, these NOES are unobservable, not because of dynamics, but because of the nature and extent of the double helix, which prevents distant parts of the molecule from folding back upon itself. The difficulty of achieving a structure without these NOES is similar to the challenge facing investigators of non-globular protein or DNA conformations. As with these other molecules, subtleties of the global structure, such as a slight bend in the helix, are beyond the resolution of the NOE or coupling constant constraints. However, the data are sufficient to determine local and medium-range structure. In this case, the data specify that the intertwined double helix is right-handed, with approximately 57 residues per turn. The helix width and length as defined in the results are near 26 A and 27 A, respectively. Table 4 compares these results with several other double helical conformations for gramicidin A. This structure is similar to the parallel helix modeled by Arseniev et al. (1986), except for length. The length is closer to that of the species 3 crystal structures (Langs, 1988; Wallace & Ravikumar, 1988) or that of the cesium complexed solution structure (Arseniev et al., 1985). The pore width is comparable with all of the uncomplexed structures. A few structural details are worth noting. Large amplitude local motions, such as rotations or flips of the tryptophan rings, do not appear to occur either slowly or rapidly with respect to the n.m.r. timescale. Slow motions would result in more than one set of resonances for several atoms, something that is not observed. Rapid motions would necessitate a series of structures with different side-chain conformations to account for the NOES. In fact, the 100 side-chain NOES are satisfied by the structure presented with no major violations, by only assuming rapid motion about methyl group symmetry axes. Also, there appears to be no significant interaction, such as stacking, between the tryptophan side-chains. This is surprising, considering that there are eight indole rings at one end of the dimer. Such a cluster of aromatic rings in a cytoplasmic protein would typically be part of a hydrophobic domain and potential nucleation site for protein folding. Other than the numerous indole

6

Backbone r.m.s.d.Q

r.m.s.d.5

for 6 structures 2.65 575

for average 2.62

in Figure

1.273 1.132 1.181 1.271 1.125 1.291

6637 0.519 0.563 0.461 0444 0592

1.212

0.52 1

minimization

t Measured as axial distance between Val’-N ethanolamine-C1. $ Average van der Waals’-van der Waals’ backbone § Calculated from differences between these structures average structure (Fig. 7).

and

1107

A Species

the

distance. and the

structure) is 6694 A. For the entire molecule, this r.m.s.d. is 1295 A, due to a relative paucity of side-chain NOES. Figure 7 shows the average of these six structures. The helix length, measured between the furthest two backbone atoms bonded to protons for which we have assigned NOES (Val’ N and the ethanolamine C-l), helix backbone to backbone width and number of residues/turn are shown in Table 3. Since an average structure necessarily has distorted bond lengths and angles, further refinement was performed on the average structure. A total of 200 cycles of minimization were followed by 100 cycles of simulated annealing with shake enabled as above, but with damping present when the penalty rose above ten. This was followed by 400 cycles of minimization. The resultant structure is visually similar to Figure 7. Statistics are shown in Tables 2 and 3.

average

4. Conclusion The structure of species 4 isolated in dioxane has been determined without the aid of long or mediumrange intramolecular NOES. Unlike many poly-

Table 4 Comparison

of n.m.r.-derived

structure for species 4 isolated in dioxane

double Environment CsSCN MeOH/CDCl, CsCl crystal Crystal Dioxane EtOH(I Dioxane

helical

Reference Arseniev et al., 1985 Wallace & Ravikumar, Langs, 1988 Arseniev et al., 1984 Arseniev et al., 1986 This study

t Valr-N to Valr-N. $ Formyl-0 to formyl-0. 5 Valr -N to ethanolamine-C-l. 11Structure predicted through

use of

a shortened

gramicidin Hand

1988

analog

r 1 1 1 1‘ 1‘

in dioxane.

with

other

structures Parallel/ antiparallel a a a a P P

Residues/ turn 7.2 64 56 56 56 57

Length (4

Width (4

27t 26 31 361 36 275

40 49 20 30 3.0 26

S. M. Pascal and T. A. Cross

1108

Figure clarity,

7. r.m.s. 1 monomer

averaged is shown

conformation as a solid

line

(stereo while

view) ofthe 6 structures from the other is shown as a broken

Fig. 6; (a) backbone; line.

(b)

all atoms.

For

Structure ?f an Isolated Gramicidin to leucine methyl NOES, the hydrophobic domain does not appear to be a major stabilizing influence in this structure. Rather, the intricate pattern of hydrogen bonding between peptide chains seems to provide the driving force for formation of this structure. The lack of NOE violations, suggesting a relatively stable structure, further suggests the possinteractions as a ibility of peptide-solvent mechanism for stabilizing the side-chain conformations. It is hoped that this and other structures of gramicidin in organic solvents will help to elucidate the nature of solvent-protein interactions. Further refinement of this conformation, including use of dihedral angle restraints, is in progress. We are deeply indebted to Hank Henricks for his help with the h.p.1.c. chromatography and Michael Nilges for discussions regarding and code-utilizing X-plor version 1.5. The n.m.r. spectrometer was purchased with the aid of NIH grant SIO RR04077-01. T.A.C. acknowledges the support of NIH grant AI-23007 and The Alfred P. Sloan foundation for a Research Fellowship. S.M.P. acknowledges the support of a University Fellowship.

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