Molecular structure of l -lysyl-l -tyrosyl-l -serine acetate

Molecular structure of L-lysyl-L-tyrosyl-L-serine a c e t a t e N. Verdaguer, I. Fita and J. A. Subirana Unidad de Quimica Macromolecular Escuela T. S. de Ingenieros Industriales, Diagonal 647, 08028 Barcelona, Spain

(Received 16 November 1989; revised 1 March 1990) The structure o f the tripeptide L-lysyl-L-tyrosyl-L-serine acetate was determined by X-ray diffraction. The crystals are triclinic space group P1, with two peptide molecules in the unit cell. The peptides are in zwitterionic form with positive charges both in the amino terminal and e-amino groups o f lysine. A negative charge is found in one o f the carboxylic groups, whereas the other one is protonated. Both peptides show very similar backbone torsional angles, in the fl pleated sheet region, but different tyrosine and serine side-chain conformations. The two lysine side chains have a similar conformation g + tg + t, which had not been previously found. In the unit cell we also find one water molecule, one isopropanol molecule and four acetic acid molecules, three o f them likely to be present as acetate anions. These molecules form layers which separate the [3-pleated sheets. The whole structure looks like an ordered solution o f peptides in the [3-sheet conformation. An extensive network o f hydrogen bonds stabilizes the crystal structure. Keywords: Crystal structure; lysine; peptide conformation;serine; tyrosine

Introduction High resolution diffraction analysis of short peptides gives detailed information that is useful in protein conformation studies. This paper is part of a project that involves the determination of the structure of different peptides with aromatic and basic side chains 1-6, sequences that are of interest in DNA binding proteins.

Experimental L-Lysyl-L-tyrosyl-L-serineacetate from Serva was crystallized by vapour diffusion of 2-propanol into a 2-propanol/acetic acid solution of the peptide. Triclinic plate-like triangular crystals appeared after several weeks. A crystal 0.25 x 0.15 x 0.06 mm was mounted in a sealed capillary. X-ray data were collected using an Enraf-Nonius CAD-4 diffractometer with CuK~ radiation ( 2 = 1.5416A) and graphite monochromator. The cell parameters were determined from 25 independent reflections (8 < 0 < 12) and are given in Table 1. The intensities of 4029 spots were measured at room temperature, using the w-scan method. Three reflections were measured every 2 h as an intensity control. Lorentz decay and polarization corrections were applied in order to derive the structure factor amplitudes, but the small effects due to absorption were ignored. The structure was determined by direct methods using the SHELXS86 computer program package 7. Cycles of least square refinement and difference Fourier syntheses done with the SHELX76 computer program 8 showed all non-hydrogen atoms and some of the hydrogen atoms in the structure. Those bonded to the O atom of the phenol ring of the tyrosine and to the O atom of the carboxylic group were placed in the positions found in the difference Fourier map and refined isotropically. 0141-8130/90/0503154)6 (t) 1990 Butterworth Heinemann Limited

Their coordinates, bond distances and angles are presented in Tables 2 and 3. The remaining hydrogen atoms were included at calculated positions and refined isotopically with geometrical constraints. The final isotropic temperature factor for calculated hydrogen atoms bonded to N1, N'I, NZ1, NZ'I was 0.0856, for those bonded to CA1, CA'l, CA2, CA'2, CA3, CA'3 was 0.0105, for those bonded to CB1, CB'I, C G I , C G ' I , CD1, CD'I, CE1, CE'I, CB2, CB'2, CB3, CB'3, was 0.0616, and for those bonded to N2, N'2, N3, N'3 was 0.0662. Anisotropic full-matrix refinements for non-hydrogen atoms (Table 2) and isotropic for hydrogens converged to a standard agreement factor R = 0.070 (R w = 0.069) for 1989 reflections with I = 3a(I) and p = 5.81 mm -1 The weighting scheme used in the last cycle was (tr2(F) + 0.0013621F12)-1. The maximum and minimum heights in the final difference Fourier map were 0.36 and

Table 1 Crystal data Space group Z a b c c~ fl 7

P1 2 9.6105 (18)/~ 12.3213 (9) A 12.4102 (12) A 96.95 ° (7) 97.19 ° (14) 99.22 ° (11)

Molecular formula (CI7H28N406)2/(CH3COO)4/CH3CHOHCH3/H20 Formula weight 533.5 Da Calculated density 1.15 g/cm 3

Int. J. Biol. Macromol., 1990, Vol. 12, October

315

Molecular structure of L-lysyl-L-tyrosyl-L-serine acetate: N. Verdaguer et al. Table 2 Fractional atomic coordinates with estimated standard deviations in parentheses and equivalent isotropic thermal parameters (A) Atom

X/A

N'I 0,1724 ( O) CA'I 0.2648 (15) CB'I 0.2572 (15) CG'I 0.1105 (19) CD'I 0.1074 (19) CE'I 0,1887 (20) NZ'I 0.1767 (13) C'I 0.2125 (14) 0'i 0.0878 (i0) N'2 0.3035 (I0) CA'2 0.2754 (15) CB'2 0.3259 (17) CG'2 0.3499 (18) CD'2 0.4842 (18) CD 2 0.2420 (17) CE'2 0.5121 (17) CE 2 0.2711 (18) CZ'2 0.4083 (20) OH'2 0.4390 (13) HOH' 0.3687 (13) C'2 0.3550 (16) 0'2 0.4780 (I0) N'3 0.2879 (12) CA'3 0.3626 (13) CB'3 0.3411 (17) 0G'3 0.4141 (II) C'3 0.3160 (16) 01'3 0.1860 (I0) 02'3 0.4031 (10) N1 0.9052 (13) CA] 0.8013 (16) cB1 0.7486 (16) cG1 0.8604 (18) col 0.7947 (20) CEI 0.7373 (22) NZI 0.6717 (16) Cl 0.8634 (18) 01 0.9930 (11) N2 0.7755 (13) C~2 0.8272 (17) CB2 0.8899 (16) CG2 0.7835 (19) C02 0.6965 (19) CD'2 0.7756 (18) CE2 0.5998 ()21 CE'2 0.6787 (25) cz2 0.5815(21) OH2 0.4859(15) HOH2 -0,5178 (15) C2 0.7117 (18) 02 0.5874 (11) N3 0.7624 (13) CA3 0.6673 (16) C83 0.6240 (19) 0G3 0.7466 (15) C3 0.7372 (17) 013 0.8673 (I0) 023 0.6582 (I0) H023 0.5523 (10) 014 0.5458 (19) OIAI 0.8995 (15) 0~I 0.8971 (14) CIAI 0.8738 (30) C2AI 0.8926 (21) OIA2 0.3325 (14) 02A2 0.4533 (15) CIA2 0.1995(24) C~2 0.3357 (26) OIA3 n.2309(14) 0~3 6.2508(15) C1A3 0.1517 (23) C2A3 0.2172(20) OIA4 0.3053(17)

316

Y/B

Z/C

BEQ

0.1736 ( O) 0.2596 (I0) 0.2180 (II) 0.1864 (14) 0.1554 (13) 0.0628 (15) 0.0263 (I0) 0.3687 (I0) 0.3677 ( 7) 0.4606 (7) 0.5730 (11) 0.6022 (12) 0.7289 (13) 0.7921 (14) 0.7831 (13) 0.9046 (13) 0.8987 (13) 0.9604 (13) 0.0709 ( 9) 1.1204 ( 9) 0.6532 (11) 0.6493 ( 7) 0.7350 ( 8) 0.8333 (10) 0.8167 (13) 0.9117 ( 8) 0.9376 (12) 0.9385 ( 7) 1.0240 (7) 0.8895 (10) 0.7921 (12) 0.7965 (12) 0.8060 (15) 0.8135 (16) 0.9186 (15) 0.9232 (12) 0.6883 (12) 0.6951 ( 8 ) 0.5974 ( 9 ) 0.4970 (11) 0.4991 (13) 0.5026 (14) 0.4127 (14) 0.6021 (15) 0.4194 (15) 0.6124 (17) 0.5196 (18) 0.5263 (II) 0.6082 (11) 0.3958 (12) 0.4038 ( 8) 0.3034 ( 9) 0.1950 (II) 1.1741 (14) 0.1727 (I0) 0.1036 (12) 0.1139 (9) 0.0180 ( 8) 1.0328 ( 8) 1.1169 (13) 0.8842 (14) 0.9424 (14) 1.0813 (17) 0.9597 (21) 0.8600 (ii) 0.7304 (10) 0.6733 (19) 0.7608 (22) 0.1867 ( 9) 0.2281 (I0) 0.3530 (15) 0.2463 (15) 0.3900 (11)

0.6967 ( O) 0.6481 (12) 0.5222 (12) 0.4624 (13) 0.3349 (14) 0,3101 (12) 0.1903 (I0) 0.6688 (11) 0.6825 ( 9) 0.6764 (8) 0.7108 (12) 0.8357 (13) 0.8702 (12) 0.8825 (13) 0.8888 (12) 0.9090 (15) 0.9147 (13) 0.9255 (14) 0.9568 (i0) 0.9152 (i0) 0.6499 (13) 0.6340 ( 8) 0.6213 ( 9) 0,5838 (ll) 0.4605 (11) 0.4217 ( 9) 0.6249 (II) 0.6265 ( 8) 0.6590 (9) 0.7021 (I0) 0.6493 (13) 0.5276 (12) 0.4522 (15) 0.3329 (14) 0.3231 (15) 0.2098 (12) 0.6648 (12) 0.6717 (10) 0.6763 ( 9 ) 0.6968 (12) 0.8139 (14) 0.8945 (15) 0.9159 (15) 0.9525 (17) 0.9867 (18) 1.0268 (15) 1.0415 (15) 1.1092 (12) 1.1552 (12) 0.6551 (12) 0.6422 ( 9) 0.6401 ( 9) 0.6065 (13) 0.4807 (15) 0.4280 (1i) 0.6457 (14) 0.6609 (10) 0.6607 (I0) 0.6728 (10) 0.1825 (13) 0.9138 (12) 1.0878 (12) 0.9637 (27) 0.9892 (20) 0.2079 (I0) 0.1771 (ii) 1.1419 (23) 0.1750 (18) 0.9187 (iI~ 1.0958 (11i 0.9853 (18) 1.0016 (19) 0,2509 (12)

2.1~ 2.40 2.54 4.25 4.6! 5.00 3.77 ].50 3.03 1.90 2.62 3.69 3.09 3.91 3.11 4.12 3.29 3.81 6.43 9.47 2,55 2.85 2.16 1.51 3.07 4.29 1.70 2.96 3.22 2.21 1.82 2.43 3.77 4.2~ 4.79 4.41

2.24 3.46 1.90 2.23 2.66 3.16 3.59 4.03 5.02 5.08 3.53 6.27 9.49 1.73 2.81 2.07 2.62 3.60 5.26 2.41 3.68 3.19 1.77 10.30 7.77 7.69 10.24 5.67 5.05 5.89 7.86 5.57 4.91 5.19 5.59 3.95 6.30

Int. J. Biol. Macromol., 1990, Vol. 12, October

0~4 CIA4 C~4 Clls C21S C3I~ O!s

0.5287 0.4847 0.4426 0.9115 0.8995 1.0458 0.8085

(19) (33) (34) (41) (39) (31) (!7)

0.3410 (13) 0.5071 (20) 0.4025 (16) 0.4146 (33) 0.4739 (30) 0.4992 (22) 0.3969 (13)

0.2871 (14) 0.3881 (19) 0.3029 (16) 0.2224 (36) 0.3236 (34) 0.4063 (24) 0.3882 (13)

8.04 9,77 5.34 16.87 14.55 11.72 9,84

-0.33 eA -3 respectively. A micro Vax 2000 computer was used for all the calculations.

Results and discussion Two peptide chains, four acetic acid molecules, one water molecule and one 2-propanol molecule are found in the unit cell.

Peptide structures The molecular structure and atomic numbering are shown in Figure I, and the final positional and thermal parameters are listed in Table 2. Bond lengths and bond angles are given in Table 3 and conformational angles (IUPAC-IUB Commission on Biochemical Nomenclature) 9 are presented in Table 4. The unit cell contains two different conformers of the tripeptide, with very similar main chain and lysine conformations, but with clear differences in the tyrosine and serine side chains. This result is not surprising, since, as recently reviewed by Karle 1°, the conformation of small linear peptides is affected by the crystallization conditions and it is common to find multiple conformations in the unit cell. The bond lengths and angles, given in Table 3, do not show any meaningful deviation from standard values. All peptide bonds show a planar conformation (~o is trans) (Table 4). Only in molecule B o~= 165.9 shows an important deviation from the standard value 09 = 180. The ~bl values observed (146 ° for chain A and 152 ° for chain B) do not differ very much from those reported in the literature for cases in which lysine is the first residue 5'6. The conformational angles (q~2, ~2) of the central residue (-153.6 °, 160.2 °) for chain A and ( - 146.0 °, 143.5 °) for chain B are close to the diagonal in the antiparallel /~-pleated sheet region in a Ramachandran map. The conformational angles of the terminal carboxyl group are close to values reported in the literatureS,6 ~b33, ~ , ~2, ( _ 152.8 o, _26.8 o, 154.0 o) for chain A and (-141.2 °, -41.8 °, 137.3 °) for chain B. In tripeptides many different structures have been described including helical and extended conformations. Thus Ala-Ala-Ala (Ref. 11), Gly-Phe-Gly (Ref. 12), Val-GlyGly (Ref. 13), Leu-Gly-Gly (Ref. 14), Lys-Ala-Ala (Ref. 5) and Lys-Tyr-Ser (this work) are examples of extended main chain conformations. Instead Ser-Gly-Gly (Ref. 15), Gly-Gly-Phe (Ref. 16), Tyr-Gly-Gly (Ref. 17), and Lys-Ala-Ala (A) (Ref. 5) are examples of an or-helical conformation. It is interesting to note that the main chains of the two crystallographically independent peptide molecules are rather accurately related by a non-crystallographic twofold axis (see Fioure 3), located between the 023 and 02'3 carboxyl oxygens of the neighbour molecules.

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Molecular structure of L-lysyl-L-tyrosyl-L-serine acetate: N. Verdaguer et al. NZ1 CD1

OG3

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OH'2

Figure 1 Conformation and atom numbering of the two independent peptides: (a) Molecule A and (b) Molecule B. For better comparison the two molecules are represented with their N-terminal groups at the right. Atoms are shown with the thermal ellipsoids of 50% probability. This and the following figures were prepared with the ORTEP program 26

Table 4 Conformational angles (°) Molecule (A)

Molecule (B)

N 1-CAI~C1 N2 CAI~I-N2~CA2 CI N2~CA2-C2 N2 C A 2 ~ 2 - N 3 CA2-C2 N3-CA3 C2-N3-CA3423 N3-CA3~C3-O 13 N3q2A3~C3~023

~kl o92 052 ~k2 o93 4)3 ~ ~,~

146.0 - 176.8 - 153.6 160.2 176.1 - 152.8 - 26.8 154.0

152.0 - 171.4 - 146.0 143.5 165.9 - 141.2 - 41.8 137.3

N2-CAI CB1-CG1 CA1-CB1-CGI~CD1 CB1 CG1-CD1-CE1 CG1 CDI~SE1-NZ1

X~ Z2 Z3 X~

59.2 - 178.1 66.6 - 178.5

52.8 174.9 57.0 175.2

N2~A2 CB2~G2 CA2~SB2~CG2~CD2 C B 2 ~ G 2 - C D 2 CE2 CG2-CD2 C E 2 ~ Z 2 CD2 CE2-CZ2~OH2

X~ Z2z Z3 Z4 Z~

65.6 82.8 - 178.3 0.9 179.0

160.1 82.1 177.6 0.0 176.8

N 3 ~ A 3 CB3~OG3

X1

60.9

180.0

before in lysine peptide structures. Finally (Z4) has been previously found in the trans and in the gauche ( - ) conformations. This result confirms our previous conclusion concerning the m a n y conformations available for the lysine side-chain. The two tyrosine side-chains show different conformations, with (Z12) =- 65.6 ° for molecule A and 160.1 ° for molecule B, they belong to one of the three most stable conformations for the side-chain of aromatic amino acids in peptides with Z12 = 60°,180 °, - 6 0 ° (Refs. 3, 4, 18-21). In all cases Z22, is close to 90 °, as found by us. The aromatic ring of the two tyrosines is planar: the biggest deviation from a plane defined by the six c a r b o n atoms is 0.038 A for molecule A and 0.025 A for molecule B. The shortest distance between tyrosine rings is 5.13 A, and discards any strong interaction a m o n g the aromatic groups in the crystal. In turn different Van der Waals interactions between the phenyl groups with both solvent and peptide molecules are observed. In particular the aromatic ring of molecule B stacks on acetate molecule A2 and is flanked by two A1 acetate molecules: the closest distances are 3.53 and 3.39 A respectively. The serine side-chains also show different conformations: (,~13) is gauche ( + ) in molecule A and trans in molecule B (Table 4). According to statistical data 18 (~1) trans, (Z 1) gauche ( + ) appear in 7% and 73% of the serine residues in peptides. However in proteins these values become 28% and 38% respectively.

Molecular packing Conformation of the side-chains As it can be seen in Figure I and in Table 4, the lysine side-chains in both molecules have very similar conformations g + , t, g + , t. Such conformation had not been previously described. As reviewed by Boqu6 et al. 6 g + (X1) and t(z 2) are frequently observed in lysine side chains of short peptides 6 but g + (Z3) had not been found

318

Int. J. Biol. Macromol., 1990, Vol. 12, O c t o b e r

Crystal packing and peptide conformations are stabilized by an extensive network of interactions where all suitable hydrogen atoms in the structure are forming hydrogen bonds (Figures 2 4 and Table 5). The main structural feature is the formation of almost fully extended antiparallel fl sheets, in agreement with the main chain conformational angles (052, ~'2) observed for both A and B peptide molecules. The side-chains do not appear

Molecular structure of L-lysyl-L-tyrosyl-L-serine acetate: N. Verdaguer et al.

P • ,2

.........

"'"A4C~ Figure 2 ORTEP plot of the hydrogen bonds (shown as broken lines) of the two peptide molecules in the cell with all the non-peptide neighbouring molecules. The molecules included in one unit cell are indicated in black; two peptides (A, B), one isopropanol (Is), four acetic acid (A1 to 4) and one water molecule (W)

FiogrUc~a4rltP aCky gho f;eh;t i~lhea~ slP:Ol;~ t ede Os~tOthe Tt~eP~anree no contacts between the side-chains, all of which interact with the acetic acid and the solvent molecules, filling the gap between the fl-sheets

Table 5

Hydrogen bond lengths (A) and angles (°)

/\ D-H . . .A

Figure 3 Crystal packing and hydrogen bonding of the peptide chains in the ab plane. Only the main chain atoms and the hydrogens of the terminal carbonyl group are indicated. The non-crystallographic twofold axes are located between 0 2 and O2'3 carboxyl oxygens and are indicated by dots

to form any h y d r o g e n b o n d with the main chain. The structure appears as a set of fl sheets immersed in a moderately h y d r o p h o b i c solvent (mainly acetic acid) which interacts with the side-chains. The fl sheets have their peptide chains in an antiparaUel arrangement and are organized 'head to tail' (type S1, $2, according to the classification of Suresh and Vijayan 22) along the b

N'I - H ... O1'3 N'I - H ...O2'3 N ' I - H ' . . . O1A3 N ' I - H " . . . O13 N1 - H ... O13 N1 - H ... 023 N1 - H ' . . . O1A1 N1 - H " . . . O1'3 NZ'I - H ... O1A2 NZ'I - H ' . . . O2A1 NZ'I - H " . . . O2A3 NZ1 - H . . . O W NZ1 - H ' . . . O2A1 NZ1 - H " . . . O2A2 N'2 - H ... 0 2 N2 - H ... 0 ' 2 O H ' 2 - H ... O1A3 OH2 - H ... O2A2 N'3 - H ... O1 N3 - H ...O'1 023 - H ... 02'3 O G ' 3 - H ... O1A2 OG3 - H ... OIs Ois - H ...O2A4 OW - H ... OH'2 OW - H ' . . . O2A4 O1A4-H ... OH2

(b) (b) (a) (c) (f) (f) (a) (g) (b) (d) (e) (a) (e) (a) (a) (a) (a) (h) (c) (g) (b) (a) (a) (a) (i) (f) (e)

H

D-A

H... A

D

2.954 3.147 2.722 2.871 2.940 3.081 2.643 2,961 2.737 2.803 2,895 2.879 2.799 2.860 2.991 3.027 2.662 2,638 2.955 3.063 2.462 2.643 a 2.844a 2.765 a 2.819 a 2.950a 3.072 a

1.882 2.388 1.690 1.974 1.960 2.185 1.650 1.892 1.733 1,749 1,882 1.814 1,730 1.817 1.944 1,996 1.665 1.573 1.903 2.023 1.407

171.2 (0.3) 169.3 (0.5) 158.1 (0.4 / 138.2 (0.3) 149.2 (1.3) 155.4 (1.21 150.3 (1.41 169.9 (1.1) 152.5 (1.3) 163.9 (1.4) 154.6 (1.3) 167.7 (1.4) 165.7 (1.5) 160.8 (1.6J 162.4 (1.1) 158.5 (1.21 147.8 (1.4) 158.9 (1.51 163.8 (1.2) 156.3 (1.3) 161.1 (1.0)

(9) (12) (14) (10) (17) (15) (20) (16) (20) (17) (19) (24) (22) (19) (16) (16) (19) (20) (16) (15) (14)

A

Symmetry code: (a) x,y,z; (b) x , y + 1,z; (c) x + 1,y,z; (d) x + l , y + l , z + l ; (e) x , y , z + 1; (f) x , y - l , z ; (g) x - l , y , z ; (h) x , y , z - l ; (i) x , y - l , z + 1 aThese H atoms are not visible in difference Fourier maps, and thei: positions can not be calculated by geometrical constraints

Int. J. Biol. Macromol., 1990, Vol. 12, O c t o b e r

31!

M o l e c u l a r s t r u c t u r e o f L - l y s y l - L - t y r o s y l - L - s e r i n e acetate." N . V e r d a g u e r et al.

axis in the crystal (see F i g u r e 3). Such i n t e r a c t i o n s t r o n g l y c o n t r i b u t e s to stabilize the fl sheet. It involves several h y d r o g e n b o n d s as s h o w n in F i g u r e 3, including the positively c h a r g e d a m i n o t e r m i n a l g r o u p s a n d b o t h c a r b o x y l t e r m i n a l g r o u p s , one of which is n o t charged. The latter c o n t r i b u t e s with its h y d r o g e n to one of the h y d r o g e n b o n d s , as s h o w n in F i g u r e 3. T w o h y d r o g e n a t o m s ( H N 1, H N ' I ) are involved in bifurcated h y d r o g e n b o n d s z3 with r a t h e r long i n t e r a t o m i c distances (see T a b l e

N.V. acknowledges a fellowship from the MEC. This work was supported by grant BT87-009.

References 1 2

5). T h e presence of an u n c h a r g e d c a r b o x y l g r o u p is r a t h e r puzzling. It interacts closely with the o t h e r c a r b o x y l group. T h e distance between the closest oxygens 0 2 3 0 2 ' 3 is 2.4/~. The p r o x i m i t y of the two c a r b o x y l g r o u p s s h o u l d increase the a p p a r e n t p K in b o t h of them, which might explain a p a r t i a l p r o t o n a t i o n of these g r o u p as it is also suggested by the presence of an electron density p e a k (0.45 e / • - 3 in the difference F o u r i e r m a p ) between the two oxygens. Therefore we have c o n c l u d e d that 0 2 3 a n d 0 2 ' 3 are likely to be forming a s t r o n g h y d r o g e n b o n d . It is interesting to m e n t i o n t h a t strong interactions between c a r b o x y l a t e g r o u p s have been described a r o u n d the 5-fold axis in S o u t h e r n Bean M o s a i c Virus (SBMV), a small i c o s a h e d r a l p l a n t virus 24 a n d between the t r a n s m e m b r a n e helices in the acetyl choline r e c e p t o r 2s. In b o t h cases the residues involved a p p e a r to be lining a gated ion channel. In o r d e r to a t t a i n a g l o b a l electrostatic b a l a n c e in the crystal, three acetic acid molecules should be ionized. The acetic acid molecules A1, A2, A3 are directly interacting with a m i n o g r o u p s and, therefore, are p r o b a b l y present as acetate anions. The r e m a i n i n g acetic acid molecule A4 a p p e a r s , in turn, to behave as a h y d r o g e n d o n o r to the h y d r o x y l g r o u p of tyrosine, which is also acting as a h y d r o g e n d o n o r . The structure of the solvent has been d e t e r m i n e d accurately from the X - r a y diffraction d a t a , b u t all molecules have a r a t h e r high mobility, as i n d i c a t e d by the values of the t h e r m a l factors ( T a b l e 2). Also some of the h y d r o g e n s d o n o t a p p e a r in the F o u r i e r difference m a p . In conclusion, a l t h o u g h m o s t a t o m s have very well defined p o s i t i o n s in the unit cell, they show s o m e mobility, so that the n e t w o r k of h y d r o g e n b o n d s m a y have some uncertainties. In a n y case the solvent molecules act s i m u l t a n e o u s l y as h y d r o g e n d o n o r s a n d a c c e p t o r s between different molecules, as shown in F i g u r e 2 a n d in T a b l e 5. T h e y certainly c o n t r i b u t e to m a i n t a i n the c o n f o r m a t i o n of the p e p t i d e side-chains a n d also the cohesion of the fl sheets.

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