Crystal Structure of the a-Helical Undecapeptide Boc-L-Ala-Aib-Ala-Aib-Ala-

Glu( OBz1)- Ala- Aib- Ala- Aib- Ala-OMe

R. BOSCH, G. JUNG, H. SCHMITT, and W. WINTER, Institut fiir Orgunische Cheniie der Universitat Tiibingen, Auf der Morgenstelle

18, D-7400 Tiibingen, Federal Republic of Germany

Synopsis

The x-ray structure of Boc-~-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala- OMe(1) represents the first a-helix determined by direct methods. This undecapeptide is a model of the N-terminus of alamethicin, and it exhibits voltage-dependent pores in bilayer membranes a t a higher voltage and concentration than alamethicin. The mol- ecule crystallizes in the monoclinic space group P2, with a = 10.602(1), b = 23.884(3), c = 13.622(1) A, p = 95.61(6)", and 2 = 2. It adopts a right-handed a-helical conformation in the solid state with intramolecular 5 - 1 hydrogen bonds. An additional intramo- lecular hydrogen bond is bifurcated, forming a stronger 4 - 1 interaction (i.e., a p-turn 111) and a weaker 5 - 1 interaction, thus prolonging the a-helical part up to 9 residues. The a-helix radius of 2.1 A, the height per res,Pue (distance N, . . . N,+$ of 1.53 A, the resulting length of the a-helical part of 13.8 A (9 residues) resp. 15.3 A (10 residues), the van der Waals radius (4.7 A), and the minimal diameter of pores formed by aggre- gation of 3-10 a-helices were calculated omitting the Glu(OBz1) side chain. In the crystal, the a-helices are linked head to tail via two hydrogen bridges forming continuous chains. Adjacent helices are oriented in antiparallel with their helix axes and have only van der Waals contacts.

INTRODUCTION

Amphiphilic helical polypeptide ionophores like alamethicin,' su- zukacillin,2 tr.ichotoxin,3 and structurally related peptides4 form volt- age-dependent pores by aggregation of a variable number of mono- meric heli~es.45~ Structural studies of a-aminoisobutyric acid* peptides in the solid state revealed a high tendency to 310-helical structures for short oligopeptides related to the antibiotic mentioned,610 and nu- merous P-turnsll were found for tri- and tetrapeptides containing a-aminoisobutyric acid. Even a decapeptide was shown to adopt a 310- helix with seven strong and one very weak intramolecular 4 + 1 hydrogen bonds.I2 Based on these results and ir and lH-nmr studies, it has been concluded repeatedly that alamethicin and other Aib-con- taining ionophores may also be mainly 310-helica1,13 and poly(Aib) is suggested to adopt a 3,,-helix according to electron diffraction studies.14

* a-Aminoisobutyric acid (= Aib)= 2-methylalanine

Biopolymers, Vol. 24, 961-978 (1985) @ 1985 John Wiley & Sons, Inc. CCC 0006-3525/85/060961-18$04.00

962 BOSCH ET AL.

The results of our crystal structure determination on the a-helical undecapeptide Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib- Ala-OMe(~)l~,’~ were in contrast to these presumptions. This undeca- peptide is related to the N-terminal helical part of alamethicin; it also exhibits membrane-modifying properties (G. Boheim and G. Jung, un- published), and it has similar nmr and CD spectroscopic properties.I6 The recently published x-ray structure of alamethicin17 confirmed our qualified choice of this a-helical undecapeptide model. In the following, full details and all relevant data of the x-ray study of I a r e given.

EXPERIMENTAL

Synthesis and spectroscopic data of the undecapeptide are published elsewhere.I6 Suitable crystals were grown from dichloromethane by addition of diethyl ether. These crystals proved to be very unstable in the absence of the mother liquor (deterioration within seconds). After numerous attempts, one crystal with dimensions of 0.22 x 0.35 x 0.48 mm3 was shown to be stable in a sealed Lindemann capillary in the presence of mother liquor. Preliminary Buerger precession pho- tographs revealed monoclinic diffraction symmetry and systematic ab- sences, according to the space group P2,. Accurate cell dimensions were obtained on a Nonius CAD-4 diffractometer, with MoK, radiation (graphite monochromator): a = 10.692(2), b = 24.22(1), c = 13.800(3) A, f i = 96.08(2)”. Intensities were collected in the 0 range of 3-25”, and after the usual data reduction, 3294 reflections with IF1 > 0 were used for further calculations (resolution -1.4 A).

Solution of the phase problem by direct methods was a troublesome and time-consuming process. In view of the large number of nonhy- drogen atoms in the asymmetric unit (90-95) and the space group P2,, an automatic solution with the programs SHELX, SHELXTL, and MULTAN 78 was obviously impossible. Additional attempts with starting reflec- tions chosen manually and the use of max. 800E-values and max. lo4 E2 relationships resulted in typical “heavy atom” solutions. Also, mod- ifications in the calculation of the E values (including molecular scat- tering curves of different molecular fragments in random orientation) failed to give sensible interpretations of the E maps.

At this stage, we decided to collect a further set of data with higher resolution. A second, larger crystal could be grown (0.40 x 0.42 x 0.43 mm3) and with CuK,, radiation, 5534 reflections with IF1 > 0 were obtained (resolution -0.9 A; for crystal data, see Table I). With this markedly improved data set and the more sophisticated programs MULTAN 80, YZARC, and MAGIC, further attempts were made to overcome the phase problem. After a year of fruitless calculations, the random- phase refinement program RANTAN (written by George Sheldrick and currently being tested in a future version, SHELX 84) led to the de- velopment of a sensible molecular fragment (50 atoms) that could be

a-HELICAL UNDECAPEPTIDE 963

TABLE I X-Ray Parameters of the Undecapeptide Z

C(52j H(94) O(16) X 3CHZC1, a = 10.602(1) 6509 reflections b = 23.884(3) c = 13.622(11 Room temperature 6 = 95.61(6) Graphite monochromator V = 3432.8 A3 2 = 2 w/&scan (8 = 3"47") Monoclinic p 2 1 M , = 1384.18

plcUc,, = 2.742 mm - l

A(CuK,) = 1.5418

5534 symmetry independent reflections

Final R value: 0.097 (R, = 0.093)

Electron density < 0.33 e/A3 in final difference for 4325 reflections with F 2 3 d F )

map

expanded to the complete molecular structure by Fourier techniques. In retrospect, the importance of the data quality (with respect to the number of observed reflections) was obvious, because the successful random-phase approach could not be repeated with the MoK, data set with reduced resolution.

During least-squares refinement (SHELX, nonhydrogen atoms an- isotropic temperature factors, solvent molecules, and hydrogen atoms isotropic), all hydrogen-containing groups were treated as rigid groups, with a fixed C-H distance of 0.96 A. Atoms O(8) and O(8)' resp. C(26) and C(26)' of the Glu(OBz1) side chain proved to be disordered, with site occupation factors of 0.5. Figure 1 shows a perspective view of I, including the atomic numbering. Tables 11-IV give the resulting atomic coordinates, bond lengths, and angles. (Lists of structure factors, ther- mal parameters, and hydrogen-atom parameters are available on re- quest.)

RESULTS AND DISCUSSION

The undecapeptide I folds into a right-handed a-helix. Values of the torsional angles (Table V) are in the region of the ideal a-helix (4 = -5T, + = -477, which is close to that of the 3,,-helix (4 = -60", $ = -30"). Deviations of the torsional angles of Ifrom the ideal values are summarized in Table VI. The hydrogen-bonding scheme of I is given in Fig. 2, in comparison with that of a p-turn (111) found in the tripeptide Boc-Gly-L-Ala-Aib-OMel8 and of a regular 3,,-helix, as observed in the pentapeptide Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe.6 The N - - - 0 distances of the six intramolecular 5 - 1 hydrogen bonds in I are collected in Table VII. The C-terminal part of the undecapeptide is completed by a 4 - 1 hydrogen bond [0(11) - - . N(10): 3.095 A] cor- responding to a &turn conformation, and finally by a 6 + 1 hydrogen bond [0(11) . - - N(11) = 3.071 A] corresponding to a r-turn. An ad- ditional interaction between O(10) and N(10) (3.116 A) is too weak to

964 BOSCH ET AL.

I

Fig. 1. Perspective view of the undecapeptide Z along its helix axis from N- to C- terminus together with the atomic numbering.

be considered as a regular 5 + 1 hydrogen bond. The geometry in this “bifurcated system” is illustrated in Fig. 3.

Recently, the angles around Ca of the Aib residues of 20 Aib-con- taining oligopeptides were examined. It was found that /3 and 819,20 should be significantly less, whereas E and 77 should be significantly greater than the value of the tetrahedral angle (109.45”) for a right- handed 3,,-helix (+ = -60”, * = -30”). In a left-handed 3,,-helix, reversed values are expected. For the denotations of the aiigles, see Fig. 4. On the other hand, an a-helical conformation should favor a symmetric geometry at Aib-Ca. Although we could confirm these ob- servations on various examples of /3-turns or 3 , , - h e l i ~ e s , ~ J ~ ~ ~ ~ - ~ ~ in the case of this a-helical compound, no symmetric geometry could be ob- served. The angles in I are compared with the average values of a survey of 20 peptides in Table VIII (see also Fig. 4).

As I represents the first a-helix determined at a much higher res- olution, as in protein crystallography, we used our coordinates to ex- amine the helix geometry. We found a value of 1.53 A for the distance N, - - - N r + 4 , divided by 4.0, which seems to be a much more charac- teristic attribute of an a-helix than the usual “rise per residue.” Ac- cordingly, the pitch of the helix has a value of 5.51 A. Thus, the length of the a-helical part of I containing 9 residues is therefore 13.8 A.

TA

BL

E I1

A

tom

ic C

oord

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es a

nd T

herm

al P

aram

eter

s of

the

Und

ecap

eptid

e N

-t-B

oc-

~-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe

x 3C

H2C

12

-0.4

049(

7)

- 0.

3154

(7)

-0.4

290(

7)

-0.2

586(

9)

0.02

54(7

) -0

.022

3(7)

-0

.058

4(7)

0.

0 158

(2

-0.0

63

(3)

-0.1

75 (1

) 0.

2160

(7)

0.39

09(7

) 0.

2660

(8)

0.31

69(8

) 0.

4973

(9)

0.45

9 (1

) 0.

376

(1)

-0.5

262(

8)

-0.5

02

(1)

-0.2

463(

9)

- 0.

1342

(9)

-0.2

014(

8)

-0.0

820(

8)

0.16

72(7

) 0.

1960

(9)

0.16

32(9

) 0.

3678

(9)

0.01

35(0

) - 0.

0097

(4)

-0.0

405(

3)

-0.1

513(

4)

-0.0

787(

4)

0.02

03(4

) -0

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1(4)

- 0.

3241

(9)

-0.2

74

(1)

- 0.

3214

6)

-0.1

498(

4)

-0.0

314(

4)

0.01

88(3

) -0

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6(4)

-0

.247

9(4)

-0

.324

4(4)

-0

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1(4)

-0

.009

7(4)

- 0.

1051

(4)

-0.1

280(

4)

-0.0

277(

4)

-0.0

258(

4)

- 0.

1227

(4)

-0.0

943(

4)

-0.0

018(

4)

- 0.

0505

(4)

- 0.

1189

(4)

-0.2

298(

5)

- 0.

0774

(5)

0.12

70(5

) 0.

1869

(6)

0.16

97(5

) 0.

3241

(5)

0.50

58(5

) 0.

271

(2)

0.15

38(9

) 0.

221

(1)

0.48

88(5

) 0.

5241

(6)

0.73

65(6

) 0.

8382

(5)

0.75

83(6

) 0.

5738

(8)

0.69

02(8

) -0

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1(6)

0.

0176

(7)

0.03

09(6

) 0.

0948

(6)

0.28

36(6

) 0.

3648

(6)

0.35

91(6

) 0.

4814

(6)

0.65

30(6

) 0.

6889

(7)

0.05

4(5)

0.

057(

5)

0.05

5(5)

0.

087(

6)

0.05

5(5)

0.

057(

5)

0.05

9(5)

0.

139(

7)

0.13

9(7)

0.

21 (

2)

0.05

7(5)

0.

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5)

0.08

4(6)

0.

074(

6)

0.09

0(7)

0.

094(

8)

0.08

7(7)

0.

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6)

0.07

1(6)

0.

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6)

0.05

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0.

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5)

0.04

0(5)

0.

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5)

0.05

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0.

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0.

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0.16

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6)

0.06

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0.06

6(5)

0.

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6)

0.08

3(7)

0.

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7)

0.05

8(6)

0.

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0.06

3(7)

0.

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7)

0.05

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0.05

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3(4)

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0.04

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0.04

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0.11

(1)

0.05

4(5)

0.

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0.05

1(5)

0.

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4)

0.07

1(6)

0.

101(

8)

0.12

6(9)

0.

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5)

0.04

6(6)

0.

037(

5)

0.03

5(5)

0.

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5)

0.04

8(5)

0.

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5)

0.03

8(5)

0.

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5)

0.05

1(5)

0.01

6(4)

0.

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4)

O.O

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(4)

O.O

lO(4

) 0.

002(

4)

-0.0

01(4

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(4)

-0.0

6 (1

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4)

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(4)

0.01

3(4)

0.

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5)

0.01

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0.

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6)

0.00

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(5)

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0.00

4(5)

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5)

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l(5)

0.00

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0.

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5)

-0.0

03(5

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01(4

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012(

4)

-0.0

07(4

) -0

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(4)

-0.0

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.015

(4)

O.O

OO

(4)

0.01

(1)

- 0.

023(

4)

-0.0

16(4

) -0

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(4)

-0.0

13(4

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0.04

0(6)

0.

033(

6)

- 0.

004(

4)

-0.0

14(5

) -0

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(4)

-0.0

03(4

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(4)

-0.0

07(4

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- 0

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(4)

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12(5

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-0.0

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- 0.

006(

4)

O.O

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(5)

0.00

6(5)

-0

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(4)

-0.0

01(5

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016(

4)

-0.0

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) M

O

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(4)

E 0

Q h K c Z

-0.0

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(4)

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5)

M

0.00

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0.

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6)

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0.01

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0.00

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0.

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5)

0.00

3(4)

-0

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(4)

- 0.

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0.00

5(5)

0.

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5)

(con

tinu

ed)

u1

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G?

G?

TAB

LE I1

(con

tinu

ed)

-

xla

y

/b

0.33

2 (1

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.332

(2

) -0

.289

(1

) -0

.234

(2

) -0

.201

(2

) -0

.405

(1

) -0

.552

(1

) -0

.694

(1

) -0

.488

(1

) -0

.454

(1)

-0.4

73

(2)

-0.5

20

(1)

-0.3

10

(1)

-0.1

11

(1)

-0.0

61

(2)

-0.0

67

(1)

-0.1

04

(1)

-0.2

12

(1)

0.02

5 (1

) -0

.103

(1

) -0

.212

(1

) -0

.341

(1)

-0.1

10

(1)

0.01

3 (1

) 0.

021

(1)

-0.0

94

(1)

-0.0

712(

4)

-0.0

722(

9)

-0.1

57

(2)

-0.2

73

(1)

-0.2

310(

4)

0.05

05(8

) 0.

0294

(7)

0.07

7 (1

) -0

.017

(7

) - 0.

0026

(5)

-0.0

072(

5)

- O

.OllO

(6)

- 0.

0533

(5)

-0.1

521(

5)

-0.2

049(

5)

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557(

6)

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422(

5)

- 0.

1232

(6)

- 0.

1206

(8)

-0.0

74

(5)

0.02

22(5

) -0

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0.

0461

(6)

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045(

5)

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414(

5)

-0.0

675(

6)

-0.0

828(

5)

- 0.

1633

(5)

-0.2

085(

5)

-0.2

456(

6)

- 0.

2904

(1)

- 0.

2895

(5)

-0.3

651(

8)

-0.3

581(

4)

0.64

78(8

) -0

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(1

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2676

(9)

-0.2

11

(1)

-0.2

71

(6)

- 0.

1360

(8)

-0.0

153(

8)

-0.0

113(

9)

0.04

87(7

) 0.

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(8)

0.02

2 (1

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171 (1)

0.10

26(9

) 0.

0444

(8)

- 0.

0529

(9)

0.10

91(8

) 0.

1544

(8)

0.13

0 (1

) 0.

132

(1)

0.26

30(8

) 0.

3829

(8)

0.39

0 (1)

0.42

29(7

) 0.

3996

(8)

0.32

25(9

) 0.

318 (1)

0.24

2 (1

) 0.

2405

(3)

0.14

8 (1

) 0.

0780

(8)

0.08

1(81

0.

09 (

1)

0.07

2(9)

0.

17 (

2)

0.11

(1)

0.

055(

7)

0.06

4(8)

0.

070(

9)

0.05

2(7)

0.

071(

8)

0.10

(1)

0.

07 (

1)

0.07

3(8)

0.

055(

7)

0.12

(1)

0.

063(

8)

0.06

7(8)

0.

079(

9)

0.06

1(8)

0.

048(

6)

0.04

1(6)

0.

065(

9)

0.05

0(6)

0.

038(

6)

0.04

7(7)

0.

068(

9)

0.13

9(8)

0.

139(

8)

0.15

(2)

0.

15 (

2)

0.05

1(7)

0.

13 (

1)

0.09

(1)

0.

14 (

2)

0.10

(1)

0.

059(

8)

0.04

7(7)

0.

074(

9)

0.06

1(8)

0.

056(

8)

0.05

1(8)

0.

08 (1)

0.04

3(7)

0.

074(

9)

0.11

(1)

0.

062(

8)

0.05

5(8)

0.

054(

8)

0.07

1(9)

0.

054(

7)

0.06

8(8)

0.

08 (

1)

0.05

9(8)

0.

050(

7)

0.05

6(8)

0.

064(

9)

0.09

(1)

0.05

0(9)

0.07

1(7)

0.

06 (

9)

0.04

9(7)

0.

09 (

1)

0.13

(1)

0.05

3(7)

0.

042(

6)

0.04

6(7)

0.

034(

5)

0.04

9(7)

0.

08 (

1)

0.07

5(9)

0.

064(

8)

0.04

7(6)

0.

047(

8)

0.03

8(6)

0.

052(

7)

0.06

3(8)

0.

075(

9)

0.04

5(6)

0.

051(

6)

0.07

0(9)

0.

039(

6)

0.05

1(6)

0.

072(

8)

0.08

1(9)

0.11

(1)

0.07

(1)

-0.0

06(6

) 0.

04 (1)

0.01

7(7)

0.

03 (1)

0.04

(1)

-0.0

02(6

) O.

OOO(

5)

0.01

1(6)

0.

004(

5)

0.00

9(6)

O

.OO

O(7

) 0.

024(

8)

-0.0

05(6

) -0

.011

(6)

-0.0

09(9

) 0.

003(

6)

0.01

1(6)

O

.OO

l(7)

0.00

9(8)

0.

006(

6 0.

005(

6)

0.00

5(8)

0.

008(

5)

0.00

5(5)

-0

.001

(7)

- 0.

013(

8)

-0.0

3 (1

) -0

.012

(8)

- 0.

020(

6)

-0.0

02(8

) O

.OO

l(7)

0.02

(1)

0.09

(1)

0.00

7 (6)

O.

OOO(

5)

0.00

6(6)

- 0.

003(

5)

-0.0

17(6

) -0

.025

(8)

-0.0

03(8

) -0

.006

(6)

0.00

2(6)

0.

017(

8)

0.00

9(5)

-0

.011

(6)

-0.0

14(7

) O

.OO

O(7

) 0.

004(

5)

0.00

9(5)

0.

007(

7)

O.OO

O(5)

-0

.007

(5)

-0.0

06(3

) O

.OO

O(7

)

-0.0

2 (1

) -0

.03

(1)

O.OO

O(6)

-0

.01

(1)

O.OO

l(8)

-0.0

7 (2

) 0.

01 (1)

O.OO

O(6)

0.

002(

6)

0.00

9(7)

O

.OO

l(6)

O.O

OO

(7)

w 0

-0.0

27(8

) C?

0.01

6(7)

3

0.00

6(7)

p

0.

011(

7)

'

0.01

6(7)

0.00

6(6)

0.

005(

6)

O.O

lO(8

) -0

.014

(6)

0.00

5(5)

0.

002(

6)

-0.0

07(7

)

-0.0

20(8

)

0.00

2(6)

z

0.01

(1)

~

- 0.

008(

7)

-0.0

2 (1

) 0.

00 (

1)

-0.2

61

(1)

-0.3

68

(1)

-0.4

88

(1)

-0.5

01

(1)

-0.3

94

(1)

0.14

5 (1

) 0.

287

(1)

0.30

3 (1

) 0.

294

(1)

0.19

1 (1

) 0.

059

(1)

0.28

7 (1

) 0.

212

(1)

0.16

5 (1

) 0.

063

(1)

0.29

5 (1

) 0.

478

(1)

0.52

8 (1

) 0.

581

(1)

0.43

7 (1

) 0.

284

(1)

0.16

6 (2

) 0.

386

(1)

0.46

1 (2

) 0.

195

(3)

0.25

8 (2

) 0.

427

(3)

0.56

0 (3

) 0.

1671

(5)

0.16

30(6

) 0.

347

(1)

0.59

2 (1

) 0.

9419

(8)

0.17

0 (2

)

-0.3

440(

4)

-0.3

384(

4)

-0.3

468(

4)

-0.3

609(

4)

- 0.

3666

(4)

- 0.

1350

(5)

-0.0

656(

5)

-0.0

289(

6)

-0.0

309(

5)

0.03

66(5

) 0.

0606

(6)

0.08

15(6

) 0.

0006

(5)

-0.0

885(

5)

- 0.

1309

(6)

- 0.

1164

(5)

- 0.

1552

(5)

- 0.

1516

(6)

-0.1

381(

6)

-0.2

157(

6)

-0.2

871(

6)

- 0.

2981

(8)

-0.3

286(

6)

- 0.

4182

(8)

0.74

7 (2

) 0.

2122

(9)

-0.2

48

(2)

-0.2

92

(1)

0.15

24(3

) 0.

2661

(3)

-0.2

783(

5)

- 0.

2573

(5)

0.22

19(4

) 0.

7272

(8)

-0.0

199(

81

-0.0

869(

8)

-0.0

559(

8)

0.04

20(8

) 0.

1090

(8)

0.42

09(8

) 0.

3751

(8)

0.28

41(9

) 0.

4671

(7)

0.56

57(8

) 0.

5630

(9)

0.57

1 (I)

0.65

96(8

) 0.

7375

(8)

0.71

51(9

) 0.

7593

(8)

0.68

89(9

) 0.

5896

(9)

0.76

9 (1

) 0.

7038

(9)

0.66

0 (1

) 0.

584

(2)

0.63

2 (1

) 0.

672

(2)

-0.0

51

(2)

0.34

6 (2

) 0.

386

(2)

0.31

3 (2

) 0.

3202

(4)

0.38

33(5

) 0.

3045

(8)

0.37

91(8

) 0.

1360

(6)

0.04

5 (1

)

0.13

(2)

0.

19 (

2)

0.19

(2)

0.

13 (

2)

0.18

(2)

0.

053(

7)

0.05

2(7)

0.

060(

8)

0.04

1(6)

0.

066(

8)

0.06

8(8)

0.

069(

9)

0.06

8(8)

0.

074(

8)

0.06

2(8)

0.

055(

7)

0.07

0(9)

0.

060(

8)

0.06

1(8)

0.

068(

8)

0.09

(1)

0.06

(1)

0.06

8(9)

0.

15 (

2)

0.28

(2)

0.

141(

8)

0.11

(1)

0.07

(1)

0.

129(

2)

0.16

2(2)

0.

245(

4)

0.24

3(4)

0.

203(

3)

0.39

4(9)

0.09

(1)

0.15

(2)

0.

07 (

1)

0.07

(1)

0.06

(1)

0.05

6(8)

0.

065(

8)

0.08

(1)

0.06

1(7)

0.

047(

7)

0.05

9(8)

0.

051(

8)

0.05

2(7)

0.

040(

7)

0.07

0(9)

0.

048(

7)

0.04

9(8)

0.

069(

9)

0.08

(1)

0.06

0(9)

0.

057(

9)

0.08

(1)

0.05

9(3)

0.

05 (

1)

0.11

(1)

0.08

(1)

0.11

(2)

0.

12 (

2)

0.10

(1)

0.04

7(6)

0.

049(

6)

0.06

1(8)

0.

042(

6)

0.05

0(7)

0.

055(

7)

0.10

(1)

0.04

0(6)

0.

041(

6)

O.Or

jO(8

) 0.

058(

7)

0.06

0(8)

0.

071(

7)

0.07

1(8)

0.

063(

8)

0.09

(1)

0.30

(3)

0.

10 (

1)

0.26

(3)

0.01

(1)

0.03

(1)

-0.0

1 (1

) -0

.03

(1)

-0.0

2 (1

) 0.

004(

6)

0.00

9(6)

0.

013(

7)

O.O

Ol(6

) 0.

006(

6)

0.00

7(6)

-0

.002

(8)

0.00

4(5)

0.

006(

5)

0.01

1(7)

-0

.005

(6)

0.00

4(6)

0.

008(

7)

-0.0

08(8

) 0.

013(

7)

-0.0

11(8

) 0.

00 (

2)

- O.

OOl(8

) 0.

03 (

1)

0.01

(1)

-0.0

1 (2

) -0

.04

(2)

-0.0

1 (1

) 0.

04 (

2)

0.00

9(5)

-0

.005

(5)

0.01

3(6)

O

.OO

l(5)

-0.0

09(6

) -0

.004

(6)

-0.0

13(8

) -0

.012

(5)

O.O

OO

(6)

0.01

3(6)

O

.OO

l(6)

-0.0

05(6

) 0.

008(

7)

-0.0

03(7

) O

.OlO

(7)

0.02

0(9)

-0

.06

(2)

-0.0

22(8

) 0.

10 (

2)

0.00

(1)

0.06

(2)

0.

03 (

1)

-0.0

2 (1

) -0

.04

(1)

- 0.

002(

6)

0.01

1(6)

-0

.006

(7)

0.00

3(6)

0.

003(

6)

~

0.01

9(7)

0.01

6(6)

O.

OlO(

6) 0

0.00

2(7)

$

-0.0

06(6

) 0.

002(

7)

0.02

1(7)

u

0.01

2(8)

M

O

.OlO

(7)

2 0.

011(

8)

+d

0.00

(1)

0.01

6(7)

4

0.03

(1)

3 M ic

-0.0

15(7

) M

Z co 3

968 BOSCH ET AL.

TABLE I11 Bond Lengths (A) with Estimated Standard Deviations in

Parentheses of the Undecapeptide

1.52(2) 1.47(3) 1.46(2) 1.43(2) 1.34(1) 1.19(1) 1.35(2) 1.42(1) 1.51(2) 1.53(2) 1.22(1) 1.31(2) 1.47(2) 1.50(2) 1.49(2) 1.54(2) 1.24(1) 1.29(2) 1.43(2) 1.48(2) 1.51(2) 1.22(1) 1.33(2) 1.46(2) 1.53(2) 1.54(2) 1.54(2) 1.19(1) 1.33( 1) 1.42(1) 1.52(2) 1.53(2) 1.21(1) 1.29(1) 1.45(1) 1.51(2) 1.50(2) 1.52(2) 1.52(2) 1.25(2) 1.28(2) 1.28(2)

1.25(2) 1.33(1) 1.33(2) 1.46(2) 1.49(2) 1.40(2) 1.40(2) 1.40(2) 1.40(2) 1.40(2) 1.40(2) 1.56(2) 1.19(1) 1.32(2) 1.44(1) 1.54(2) 1.50(2) 1.23( 1) 1.33(2) 1.42(2) 1.52(2) 1.47(2) 1.54(2) 1.22(1) 1.33(2) 1.46(1) 1.49(2) 1.53(2) 1.22(1) 1.29(2) 1.45(2) 1.50(2) 1.53(2) 1.53(2) 1.21(2) 1.3412) 1.45(2) 1.57(2) 1.54(2) 1.17(2) 1.33(2) 1.45(2)

The helix radius was calculated by averaging the maximum dis- tances of the backbone atoms from 8 least-square planes represented by the planar peptide units. The value we found (2.1 A) is significantly less than the literature value (2.3 Using the van der Waals radii given by Bondi,26 we found graphically an overall diameter of the a- helical rod of 9.4 A (Fig. 5). The next step was to calculate the thickness of pores formed by parallel aggregation of a variable number of helical

a-HELICAL UNDECAPEPTIDE

TABLE IV Bond Angles (deg) with Estihated Standard Deviations (in Parentheses)

of the Undecapeptide

108(1) 111(1) 112(1) 103(1) 109(1) 113(1) 120.3(9) 127(1) 109.2(9) 118.5(9) 108.7(9) 113.4(9) 109(1) 119(1) 117.6(9) 120.6(9) 108(1) 111(1) 106(1) l l O ( 1 ) 108(1) 113(1) 120(1) 118(1) 121.1(9) 109(1) 113(1) 112(1) 121(1) 115.3(9) 121.1(9) 107.0(9) l l O ( 1 ) 107(1) 112(1) 109(1) 11 1.8(9) 122(1) 114.8(9) 118.8(9) 108.8(9) 113.2(9) 109(1) 120(1) 117.1(9) 119.2(9) 109.4(9) 110.9(9) 109.5(9) 111(1) 105(1) 114(2) 117(2)

111(1) 106(1) 95(21

113(1) 101(1) 119(1) 120(1) 120(1) 120(1) 120(1) 120(1) 121(1) 120(1) 120(1) 114.3(9) 117.4(8) 108.4(9) 111.4(9) 111(1) 120(1) 118.0(9) 123.4(9) 107.8(9) 113(1) 109.4(9) 111(1) 107(1) 109(1) 122(1) 115.8(9) 122.8(9) 107.8(9) 111(1) 111(1) 118(1) 118(1) 125(1) 109(1) 111(1) 109(1) l l O ( 1 ) 108(1) 111(1) 123(1) 115(1) 118(1) l l O ( 1 ) 108(1) 105(1) 130(1) 106(1) 114(1)

970 BOSCH ET AL.

TABLE V Torsional Angles’ of the Undecapeptide N-t-Boc-LAla-Aib-Ala-Aib-Ala-

Glu(OBz1)-Ala- Aib-Ala-Aib-Ala-OMe

9 J, w

1 Ala 2 Aib 3 Ala 4 Aib 5 Ala 6 Glu 7 Ala 8 Aib 9 Ala 10 Aib 11 Ala

-63.9 -57.6 - 68.2 - 55.9 - 68.5 -63.7 -68.0 -55.1 - 78.1

55.7 - 56.5

-49.4 -47.1 -41.5 - 45.3 -39.6 -38.3 -47.8 -36.0 -21.3

44.7 146.6

- 159.1 - 176.5 - 174.3

178.3 180.0 178.1 180.0 179.0

- 175.1 - 164.0

180.0

For deviations from the ideal +/+-values of a- and 3,,-helices (-57”l -47” and -6W/ -3W, resp.) see Fig. 2(a) in the following paper and Table VI.

rods (Fig. 6). The values of different pore diameters for 3-10 monomers are given in Table IX. It is known that the higher conductance levels of a single pore of natural antibiotics are nonselective, as observed experimentally, e.g., by Boheim et al.27

In the examples of Fig. 6, obviously only the smallest pore, consisting of three undecapeptide monomers (which represents the lowest con- ductance level), should be ion selective. With a diameter of 1.5 b;, it should be impermeable, at least to large anions-for example, C1- (ionic radius, 1.81 A); in contrast, all larger pores should be permeable even to very large ions. However, the real diameter of such a pore remains an open question as long as the participation of water and

TABLE VI Deviations A (deg) of the Torsional Angles of Undecapeptide Z from the

Corresponding Angles of Ideal a- and 3,,,-Helices

A a Values for A310 Values for

9 Boc-’ Ala-

2Aib-

‘Aib-

6Glu(OBzl)-

8Aib-

‘OAib-

3 ~ i a -

5 ~ i a -

7 ~ i a -

9 ~ i a

Ala-OMe

- 6.9 -0.6

-11.2 +1.1

-11.5 -6.7

-11.0 + 1.9

-21.1 +112.7

+0.5

+ -2.4 -0.1 +5.5 +1.7 +7.4 +8.7 -0.8

+11.0 +25.7 c97.7 - 13.6

w

+20.9 +3.5 +5.7 - 1.7

0.0 - 1.9

0.0 -1.0 +4.9

+ 16.0 0.0

9 -3.9 +2.4 -8.2 +4.1 -8.5 -3.7 -8.0 +4.9

- 18.1 +115.7

+3.5

J, w

-19.4 +20.9 -17.1 +3.5 -11.5 +5.7 -15.3 -1.7

-9.6 0.0 -8.3 -1.9 - 17.8 0.0

-6.0 -1.0 i8.7 +4.9

174.7 +16.0 + 176.6 0.0

a-HELICAL UNDECAPEPTIDE 971

10

9

8

7

6

5

L

3

2

1

f l - turnlIII l 3,,--hel~x OC- hellx L -1 L -1 5 -1

Fig. 2. Hydrogen-bonding scheme of the tripeptide Boc-Gly-L-Ala-Aib-OMelH (p-turn 111) and the pentapeptide Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe6 (3i0 - helix) compared with the hydrogen-bonding system of the a-helical undecapeptide I.

TABLE VII Lengths of Hydrogen Bridges in the Undecapeptide

Bridge Length

(A)

Mean value [excluded O(10). . . "lo)] Mean value [included O(10). . . N(10)] Literature value (Ref. 25)

2.915 3.080 2.993 2.880 3.028 3.003 3.116

2.983 3.002

2.9 f 0.1

972 BOSCH ET AL.

k-312 -4

+219 -4 I7 6

k-310 4 Fig. 3. Illustration of the geometrical situation in the “bifurcated” hydrogen-bonding

system at N(10).

lipid molecules in the conducting pore state cannot be clarified. In the crystal state, the undecapeptide molecules are linked head-

to-tail to linear chains by two intermolecular hydrogen bonds N(1) - - - O(12)‘ of 2.921 A (symmetry code: -1 + x,y, -1 + z ) and N(2) . - . O(13)’ of 3.075 A (symmetry code: - 1 +z,y, - 1 + 2). This head- to-tail arrangement of a-helices results in linear chains, and these chains are arranged antiparallel with respect to their helix axis (Fig. 7). Between neighboring chains of helices only hydrophobic contacts are found. Three molecules of dichloromethane per asymmetric unit fill the space between the 6Glu(OBzl) side chain and the nonhelical C-terminus (Fig. 81, and they are not involved in the hydrogen-bonding system.

The antiparallel arrangement is also the most likely arrangement in a lipid bilayer. Due to the high dipole moment of the a-helix (about 3.5 Debye/peptide unit),28 the application of an electric field leads to a parallel arrangement and opening of a pore according to the flip- flop gating modeL5 Because the length of one undecapeptide molecule cannot span the bilayer membrane, two rings of head-to-tail linked

Fig. 4. Denotation of angles in an Aib-residue.

TA

BL

E V

III

Ang

les

(deg

) ar

ound

Aib

-Ca i

n a-

Hel

ical

Und

ecap

eptid

e Z

and

Com

pari

son

wit

h O

ther

Aib

Pep

tides

' Q ic

a

P Y

8 c-

77 t

P G-

wM

c

Aib

Z

121.

0 11

0.2

106.

6 10

9.6

112.

4 11

0.6

107.

5 11

6.6

124.

2 11

9.1 0

Aib

' a-

Hel

ix

119.

8 10

6.4

111.

5 10

8.2

113.

2 10

9.9

107.

5 11

5.8

125.

0 11

9.2

$ c A

ibR

12

1.9

104.

4 11

2.7

107.

8 11

0.0

111.

9 10

9.9

116.

1 12

2.3

121.

2 A

ib'O

p-

Tur

n I1

1 12

2.8

113.

7 10

6.8

107.

6 10

5.8

109.

8 11

2.7

111.

2 12

1.2

127.

6

119.

8 M

0

A

vera

ge v

alue

of

a-he

lica

l 12

0.9

107.

0 11

0.3

108.

5 11

1.9

110.

8 10

8.3

116.

2 12

3.8

Ave

rage

val

ues

of 2

0 A

ib r

es-

122.

2 10

6.8

110.

6 10

6.8

110.

6 11

0.6

111.

1 11

6.8

122.

6 12

0.4

% 3 pa

rt

idue

s in

310

-hel

ical

conf

orm

a-

tion

(R

ef. 2

0)

!

s M

For

defi

nitio

n of

ang

les,

see

Fig

. 4.

974 BOSCH ET AL.

Fig. 5. chain is ground).

Van der Waals plot of I along its helix axis. The voluminous Glu(OBz1) side omitted. The Boc-group is directed towards the viewer (C-terminus in back- The circle illustrates the overall volume of the a-helical rod.

\/--

Van der Waals plot of I along its helix axis. The voluminous Glu(OBz1) side omitted. The Boc-group is directed towards the viewer (C-terminus in back- The circle illustrates the overall volume of the a-helical rod.

Fig. 6. Illustration of pores resulting by parallel arrangement of three to six a- helical rods to regular polygons (see Table VIII).

a-HELICAL UNDECAPEPTIDE 975

TABLE IX Calculated Minimal Pore Sizes of Parallel Assemblies

of the a-Helical Undecapeptide P

Oligomers 3 4 5 6 7 8 9 10

Pore size (A) 1.5 3.9 6.6 9.4 12.3 15.2 18.0 21.1 ~~

a Including only Ala and Aib methyl groups (cf. Figs. 5 and 6).

helical rods are required to change their orientation simultaneously. It is reasonable that such an arrangement is less stable than that of the natural antibiotics. Additional evidence for head-to-tail linked a-helices comes from dielectric dispersion measurements of octanol/ dioxane solutions of a l a m e t h i ~ i n , ~ ~ trichotoxin A40,30 and the helical, membrane-pore-forming polypeptides Boc-L-(Ala-Aib-Ala-Aib-Ala),- OMe (n = 2 and 4).31 Interpretation of the observed data is only pos- sible under the assumption of head-to-tail linked dimeric rods, giving rise to high dipole moments. Finally, it should be emphasized that the C-terminal protected nonapeptide, with the natural sequence of alamethicin F30, also crystallizes in form of head-to-tail linked a / 3 ,,-helices. 24

Fig. 7. Perspective view of the antiparallel, linear arrangement of tail fashion in the crystal. Dipole vectors are represented by arrows.

I in a head-to-

Fig.

8.

Ster

eo v

iew

of

the

crys

tal a

rran

gem

ent

of I

alo

ng t

he f

ourf

old

helix

axi

s. F

or r

easo

ns o

f cl

arit

y, th

e C

H,C

l, so

lven

t m

olec

ules

, whi

ch f

ill

the

spac

e be

twee

n th

e G

lu(O

Bz1

)-si

de c

hain

and

C-t

erm

inus

, are

om

itted

.

a-HELICAL UNDECAPEPTIDE 977

The nonamphiphilic undecapeptide discussed above and the oligo- mers (Boc-L-Ala-Aib-Ala-Aib-Ala),-OMe (n = 1 4 ) without polar side chains constitute synthetic pore formers that demonstrate the essen- tial and unique role of helical arrangements of peptide chains in po- tential-dependent ion motions.3234

Note added in proofi Recently, the electrostatic interactions between the a-helix dipoles in the crystals of I were calculated by using our coordinates. The electrostatic interaction energy between one helix dipole and its 26 nearest neighbors was found to be -23 kcal mol-' [Hol, W. G. J. & De Maeyer, M. C. H. (1984) Biopolymers 23,809-8171.

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Received June 20, 1984 Accepted November 16, 1984