Benzyloxycarbonyl-L-alanyl-o-phenylalanyl-L-proline monohydrate, C25H29N3O6.H2O, crystallizes in the orthorhombic space group P212121 with four molecules in a unit cell of dimensions a = 9.594 (9), b = 9.705 (4) and c = 27.9 17 (12) &#197;. The structure has been refined to an R value of 0.067 for 2046 observed reflections. All the peptide units in the molecule are trans and the prolyl residue is in the conformation. The lone water molecule in the structure is hydrogen bonded to the carbonyl O atom in the C2 - C&#947; - exo - C&#946; endo conformation. The lone water molecule in the structure is hydrogen bonded to the carbonyl O atom in the benzyloxycarbonyl group and to one of the O atoms in the terminal carboxyl group. This internal water bridge, observed for the first time in a linear peptide, provides a model for water-mediated chain-reversal. An interesting feature of the crystal structure is the presence of an antiparallel sheet involving the alanyl and the phenyl-alanyl residues

Balaram, P.

Nagaraj, R.

Nair, C. M. K.

Ramaprasad, S.

Vijayan, M.

English

Publications of the IAS Fellows

Internal Water Bridge and Antiparallel Sheet in the Structure of 
Benzyloxycarbonyl-L-alanyl-D-phenylalanyl-L-proline Monohydrate 
BY C. M. K. NAIR, R. NAGARAJ, S. RAMAPRASAD, P. BALARAM AND M. VIJAYAN 
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 5600 12, India 
Abstract 
Benzyloxycarbonyl-L-alanyl-o-phenylalanyl-L-proline 
monohydrate, C2,H2,N30,. H 2 0 ,  crystallizes in the 
orthorhombic space group P2,2,2, with four molecules 
in a unit cell of dimensions a = 9.594 (9), b = 9.705 (4) 
and c = 27.9 17 (12) A. The structure has been refined 
to  an R value of 0.067 for 2046 observed reflections. 
All the peptide units in the molecule are trans and the 
prolyl residue is in the C,-CY-exo-CP-endo con- 
formation. The lone water molecule in the structure is 
hydrogen bonded to the carbonyl 0 atom in the 
benzyloxycarbonyl group and to one of the 0 atoms in 
the terminal carboxyl group. This internal water bridge, 
observed for the first time in a linear peptide, provides a 
model for water-mediated chain-reversal. An interesting 
feature of the crystal structure is the presence of an 
antiparallel sheet involving the alanyl and the phenyl- 
alanyl residues. 
Introduction 
Structural studies on small proline-containing peptides 
with all L, and mixed L and D sequences are valuable in 
elucidating the role of prolyl residues in determining the 
conformation of proteins, polypeptides and peptide 
antibiotics. We report here the crystal structure of the 
monohydrate of benzyloxycarbonyl-L-alanyl-D-phenyl- 
alanyl-L-proline, a proline-containing tripeptide with 
LDL sequence. In view of the importance of bound 
water molecules in the structure and function of 
proteins, it was also of interest to investigate the 
influence of the lone water molecule in the structure on 
peptide conformation and crystal packing. 
Experimental 
The compound was crystallized from an aqueous meth- 
anol solution. The space group and the unit-cell dimen- 
sions were determined from X-ray diffraction photo- 
graphs. The cell parameters were subsequently refined 
on a CAD-4 four-circle diffractometer. The density of 
the samples was measured by flotation in aqueous 
potassium iodide solution. The observed density 
[la260 ( 5 )  Mg m-31 agrees well with that (1.262 Mg 
m-3) calculated for four peptide molecules and four 
water molecules in the unit cell. 
Intensity data were collected from a specimen of 
dimensions 0 .7  x 0.4 x 0.3 mm on a computer- 
controlled CAD-4 diffractometer employing the co-28 
scan up to a maximum Bragg angle of 75" using 
graphite-monochromated Cu Ktr radiation. Of the 2582 
reflections collected in this range, 2046 having I > 
20( I )  were used for structure determination and 
refinement. The intensities were corrected for Lorentz 
and polarization factors but not for absorption ( p  = 
0.78 mm-' for Cu Ktr radiation). 
Structure analysis 
Although the structure contained only 35 crystallo- 
graphically independent non-hydrogen atoms and the 
space group was favourable, structure solution using 
direct methods turned out to be somewhat difficult. 
Attempts at phase determination employing MUL TAN 
(Germain, Main & Woolfson, 1971) using normal E 
values as well as those normalized separately in 
different parity groups and different ranges of Bragg 
angle were of no avail. E values were then calculated 
using group scattering factors for the two six-membered 
rings and the pyrrolidine ring in the structure. A 
chemically sensible fragment containing 1 2 atoms could 
be identified with difficulty in the E map corresponding 
to the best set of phases produced by MULTAN using 
these E values. This fragment was input with random 
orientation and random position in the next MULTAN 
(1977 version) run. The subsequent E map based on the 
best set of phases revealed the positions of 29 out of the 
35 non-hydrogen atoms. The rest of the atoms were 
located from a subsequent difference Fourier map. 
The structure was refined by the block-diagonal 
SFLS method using the locally modified version of a 
program originally written by Professor R. Shiono. The 
heavy atoms and the H atoms, located from a 
difference Fourier map, were assigned anisotropic and 
isotropic thermal parameters respectively in the final 
cycles. The refinement converged at R = 0.067. The 
weighting function used had the form l / ( a  + bF, + 
cFZ) where a = 1 -20, b = 0-049 and c = -0.000 1. The 
scattering factors of the non-hydrogen atoms and the H 
atoms were taken from Cromer & Waber (1965) and 
Stewart, Davidson & Simpson ( 1965) respectively. 
The computations were carried out first on an IBM 
360/44 machine and subsequently on a DEC-1090 
computer. The final positional parameters and the 
equivalent isotropic temperature factors (Hamilton, 
1959) of the non-hydrogen atoms are given in Table l.* 
* Lists of structure factors, thermal parameters and H atom 
parameters have been deposited with the British Library Lending 
Division as Supplementary Publication No. SUP 35734 (18 pp.). 
Copies may be obtained through The Executive Secretary. 
International Union of Crystallography. 5 Abbey Square. Chester 
C H  1 2HU, England. 
Table 1. Fractional coordinates ( X  1 04) and equiualent 
isotropic temperature factors (Hamilton, 1959) of the 
non-hydrogen atoms 
Standard deviations are given in parentheses. 
X 
4669 ( 1  1) 
4934 ( 1  I )  
5971 (13) 
6843 (14) 
6588 (1 2) 
5482 (9) 
5249 (13) 
5452 (6) 
4587 (7) 
3680 ( 5 )  
4878 (5) 
4131 (6) 
4139 (9) 
4821 (6) 
6088 (4) 
3951 (5) 
4456 (6) 
4343 (7) 
4977 (8) 
4197 (9) 
4779 (14) 
6136 (13) 
6863 ( 1  1) 
63 13 (9) 
3594 (6) 
2321 (4) 
4223 (5) 
3386 (7) 
4484 (10) 
5702 ( 1  I )  
571 1 (7) 
2319 (7) 
1299 (6) 
2441 (5) 
y 
471 (13) 
875 (14) 
1692 (13) 
2205 (13) 
1804 (12) 
945 (9) 
502 (10) 
1696 (7) 
1794 (8) 
973 (6) 
2943 (6) 
3263 (8) 
4792 (8) 
2488 (8) 
2342 (7) 
2093 (6) 
1459 (7) 
2490 (8) 
1918 (9) 
999 ( 1  1) 
482 (13) 
890 (15) 
1729 (17) 
2239 (12) 
182 (6) 
303 (5) 
-1027 (6) 
-2212 (7) 
-3351 (9) 
-2897 ( 1  1) 
-1320 (8) 
-2609 (7) 
-3292 (6) 
-2357 (6) 
z 
2699 (3) 
3 168 (3) 
3274 (3) 
2930 (3) 
2453 (3) 
2331 (3) 
1821 (3) 
1514 (2) 
1126 (2) 
1039 (2) 
882 (2) 
445 (2) 
349 (3) 
19 ( 2 )  
- 1  (2) 
-324 (2) 
-765 (2) 
- 1 189 (2) 
- 1934 (3) 
- 1644 (2) 
-2362 (3) 
-2487 (3) 
-2195 (4) 
- 1776 (3) 
-868 (2) 
-927 (2) 
-9 12 (2) 
-1055 (2) 
- 1  24 (4) 
-859 (5) 
-843 (3) 
-670 (3) 
-862 (2) 
-250 (2) 
4270 (6) -908 (6) 324 (2) 
B (A2) 
9.2 (5) 
9.0 (4) 
9.1 (4) 
9.8 ( 5 )  
8.5 (4) 
5.6 (3) 
8.4 (4) 
6.5 (2) 
4.7 (2) 
5.8 (2) 
4.3 (2) 
4.0 (2) 
5.7 (3) 
3.8 (2) 
5.8 (2) 
3.8 (2) 
3.5 (2) 
4.5 (2) 
4.8 (2) 
6.4 (3) 
9.4 (5) 
1 1 . 1  (5) 
10.5 (5) 
7.0 (4) 
3.4 (2) 
4 - 5  (2) 
3.8 (2) 
4.5 (2) 
7.5 (4) 
1 1.0 (5) 
5.0 (3) 
4.4 (3) 
6.6 (2) 
5.5 (2) 
7.1 (2) 
Discussion 
Bond lengths and angles 
The bond lengths and the valency angles in the 
structure, given in Table 2, are by and large close to the 
respective standard values (Vijayan, 1976). A few 
bonds in the phenyl and the pyrrolidine rings, however, 
are abnormally short. All these bonds are associated 
with atoms having high thermal parameters. The 
shortening of the Cp-Cv bond in the prolyl residue, a 
feature also observed in several other crystal structures 
(e.g. Kartha, Ashida & Kakudo, 1974; Karle, 1974), is 
of particular interest as it appears to be related to the 
conformational flexibility, albeit limited, of the pyr- 
rolidine ring. Most of the conformational possibilities of 
the ring can be described as arising from the dis- 
placement of Cp, C y  or both from the plane defined by 
the rest of the atoms (Ashida & Kakudo, 1974). These 
displacements can assume a continuous range of values 
thus giving rise to the possibility of static disorder in a 
direction perpendicular to the plane of the ring. This 
disorder, along with thermal vibrations, could lead to 
highly anisotropic temperature factors for Cp and Cy, 
and a consequent shortening of the Cp-Cv bond. In the 
present structure, the B values along the principal axes 
of the thermal-vibration ellipsoid of Cy are 20.2, 8 - 6  
and 4.2 A2; the corresponding values for Cp are 10.8, 
8.3 and 3.3 A2. In both cases, the major axis is nearly 
perpendicular to the mean plane of the five-membered 
ring, the angle between the major axis and the plane 
normal being 4.6 and 24-2" for Cy and Cp respectively. 
The C p-Cv length corrected for the temperature factor 
is 1.499 and 1.582 8, when the vibrations are approxi- 
mated to 'riding motion' and 'uncorrelated motion' 
respectively (Busing & Levy, 1964). Although neither 
approximation corresponds to the real situation, the 
values do indicate that the shortening of the bond can 
indeed be explained in terms of the appropriate thermal 
parameters. 
S ide-c ha in con f o  rm a t io n 
The prolyl residue assumes a C2-CY-ex+CP-endo 
conformation (Ashida & Kakudo, 1974) with C y  and 
Cp deviating by -0.29 1 and 0.124 A respectively from 
the plane defined by N, C a  and C8. The conformation 
of the phenylalanine side chain is defined by two 
dihedral angles x' and x21 (IUPAC-IUB Commission 
on Biochemical Nomenclature, 1970). The observed 
value of 174.9O for x1 in the structure corresponds to 
the second most favourable conformation sterically 
with the phenyl ring trans to the N H  group and gauche 
to the C O  group. The observed value of xZ1 (8 1.4") is in 
the neighbourhood of 90°, as is the case with most of 
the aromatic side chains in peptides and proteins 
(Benedetti, 1977; Bhat, Sasisekharan & Vijayan, 1979). 
Table 2. Bond lengths (A) and bond angles (") in- 
volving non-hydrogen atoms 
Standard deviations are given in parentheses. (See Fig. 1 for atom 
numbering.) 
C( 1)-C(2) 1 *39 1 (14) 
C(2)-C(3) 1.306 (17) 
C(4)-C(5) 1.409 (14) 
C(6)-C(7) 1.503 (12) 
0(8)-C(9) 1.367 (8) 
C(9)-N(ll) 1.336 (9) 
C(12)-C(13) 1.508 (11) 
C(14)-O(15) 1.226 (7) 
N(16)-C(17) 1.460 (8) 
C(17)-C(25) 1.517 (9) 
C(19)-C(20) 1.418 (12) 
C(20)-C(21) 1.410 (13) 
C(22)-C(23) 1.346 (18) 
C(25)-O(26) 1.238 (7) 
N(27)-C(28) 1 *459 (9) 
C(28)-C(32) 1 *534 (10) 
C(30)-C(3 1) 1.53 1 (1 3) 
C(32)-O(34) 1.205 (8) 
C(2)-C(l)-C(6) 120 (1) 
C(l)-C(2)-C(3) 122 (1) 
C(2)-C(3)-C(4) 122 (1) 
C(3)-C(4)-C(5) 1 17 (1) 
C(4)-C(5)-C(6) 122 (1) 
C( I)-C(6)-C(5) 116.9 (9) 
C( l)-C(6)-C(7) 122.0 (8) 
C(5)-C(6)-C(7) 121.1 (8) 
C(6)-C(7)-0(8) 108.1 (8) 
C(7)-0(8)-C(9) 116.2 (6) 
0(8)-C(9)-0(10) 123.6 (7) 
0(8)-C(9)-N( 11) 109.6 (6) 
0(10)-C(9)-N(ll) 126-8 (7) 
C(9)-N(lI)-C(12) 120.4 (6) 
N( 1 1 )-C ( 1 2)-C ( 1 3) 1 1 1 * 1 (6) 
N( 1 1)-C( 12)-C( 14) 109.2 ( 5 )  
C(13)-C(12)-C(14) 109.7 ( 5 )  
C( 12)-C( 14)-O( 15) 120.9 (6) 
C(12)-C(14)-N(16) 115.1 (6) 
0(15)-C(14)-N(16) 123.8 (6) 
C(14)-N(16)-C( 17) 121 - 5  ( 5 )  
N ( 1 6)-C ( 1 7)-C ( 1 8) 1 1 0 * 4 (5) 
N( 16)-C( 17)-C(25) 108.9 ( 5 )  
C(18)-C(17)-C(25) 110.1 ( 5 )  
C ( 1 )-C (6) 
C(3)-C(4) 
C(5)-C(6) 
C(7)-0(8) 
C(9)-O(10) 
N(ll)-C(12) 
C ( 1 2)- C ( 1 4) 
C( 14)-N( 16) 
C( 17)-C( 18) 
C( 18)-C( 19) 
C( 19)-C(24) 
C(2 1)-C(22) 
C (23)-C (24) 
O( 25)-N (2 7) 
C (28)-C (29) 
C (2 9)- C (30) 
C (3 2)-O( 3 3) 
1.369 (13) 
1.368 (16) 
1.392 (14) 
1.455 (1 1) 
1 *204 (9) 
1 *448 (8) 
1.555 (9) 
1.327 (8) 
1.553 (9) 
1.516 (10) 
1.370 (1 1 )  
1.405 (1 8) 
1.375 (14) 
1 *324 (8) 
1 *539 (1 2) 
1.451 (16) 
1 *298 (9) 
C(17)-C(18)-C(19) 112.0 (6) 
C(18)-C(19)-C(20) 119.9 (7) 
C( 18)-C( 19)-C(24) 12 1 - 1 (7) 
C(2O)-C(19)-C(24) 118.9 (8) 
C(19)-C(2O)-C(21) 119.9 (8) 
C(2O)-C(21)-C(22) 118.5 (9) 
C(21)-C(22)-C(23) 120 (1) 
C(22)-C(23)-C(24) I22 (1) 
C(23)-C(24)-C(25) 120 (1) 
C(17)-C(25)-0(26) 119.0 (5) 
C( 17)-C(25)-N(27) I 19.5 ( 5 )  
0(26)-C(25)-N(27) 121.4 (6) 
C(25)-N(27)-C(28) 118.2 ( 5 )  
C(25)-N(27)-C(3 1) 127.0 ( 5 )  
C(28)-N(27)-C(31) 114.7 ( 5 )  
N (2 7)-C (28)-C (29) 102 9 (6) 
N(27)-C(28)-C(32) 11 1.8 ( 5 )  
C (2 8)-C (29)-C (30) 1 05 * 7 (8) 
C(29)-C(3O)-C(31) 108.9 (9) 
N(27)-C(3 1)-C(30) 100.6 (7) 
C(28)-C(32)-0(33) 110.0 (6) 
C(28)-C(32)-0(34) 124.6 (6) 
O(3 3)-C (32)-O(34) 125 * 3 (6) 
Backbone conformation and the intramolecular water 
bridge 
A perspective view of the molecule is shown in Fig. 1. 
The lone water molecule in the structure, which bridges 
the two ends of the molecule through hydrogen bonds 
with the carbonyl 0 in the benzyloxycarbonyl group 
and one of the 0 atoms in the terminal carboxyl group, 
is also shown in the figure. The dihedral angles which 
define the main-chain conformation (IUPAC-IUB 
Commission on Biochemical Nomenclature, 1970) are 
listed in Table 3. All the peptide groups in the molecule 
are trans and nearly planar with 5.7O as the maximum 
value of dw. The q, w values of the prolyl residue may 
be considered as corresponding to those for the 
Fig. 1. A perspective view of the molecule. The internal water 
bridge is also shown. 
Table 3. Main-chain torsion angles (O) 
E.s.d.3 are ca 0.6O. 
i =  1 i =  2 i = 3  
W1 174.7 -174.3 
qi -83.6 132.6 -66.9 
Wi 147.0 -122.5 I57*1* 
* This torsion angle is defined by the atoms N(27), C(28), C(32) 
and O(33). 
polyproline I1 structure whereas those of the phenyl- 
alanyl residue are those appropriate for the p structure. 
In spite of its participation in an antiparallel sheet in the 
crystal structure (see below), the alanyl residue has p, 
tp values substantially different from those for an ideal 
p structure, presumably on account of the distortions 
introduced by the formation of the internal water 
bridge. 
Geometrical features of the hydration of peptides in 
crystal structures have been studied recently by Yang, 
Brown & Kopple (1979). This study indicates that 
intramolecular water bridges have been observed so far 
only in cyclic peptides [even in cyclic peptides, the 
water molecule appears to play an important role in 
determining peptide conformation only in antamanide 
(Karle, 1977)l. Thus the present structure constitutes 
the first example of an internal water bridge in a linear 
peptide. The role of the water molecule here appears to 
be the initiation of chain-reversal, which is often 
achieved in peptide chains through the formation of a 
b-turn stabilized by a 4+1 hydrogen bond. 
Bound water molecules are believed to play an 
important role in maintaining the structural integrity of 
proteins, probably by contributing to the local 
stabilization of conformation (Finney, 1977). It has 
been noticed in protein structures that more water 
molecules bind to the main chain than to side chains. 
Although the main chain has an equal number of CO 
and NH groups, more water molecules are known to 
bind to the former than to the latter in proteins (Finney, 
1977) as well as in small peptides (Yang et al., 1979). 
Thus the main-chain CO groups are the most important 
loci for water binding in polypeptide chains. In this 
context, the present structure provides a possible model, 
at atomic resolution, of a structurally important 
intramolecular water bridge involving two main-chain 
CO groups. 
Crystal structure and hydrogen bonding 
The arrangement of the molecules in the crystal 
structure is shown in Fig. 2. The parameters of the 
hydrogen bonds, which along with van der Waals 
interactions stabilize the crystal structure, are given in 
Table 4. An interesting feature of the structure is the 
presence of an antiparallel hydrogen-bonded sheet 
involving the alanyl and the phenylalanyl residues. This 
sheet extends over the entire crystal along the direction 
of the hydrogen bonds. The only other crystal struc- 
ture in which such an arrangement has been observed 
appears to be that of trialanine (Fawcett, Camerman & 
Camerman, 1975). The sheet accounts for two 
crystallographically independent N-H a 0  hydrogen 
bonds. In addition, there are three 0-Ha - a 0  hydro- 
gen bonds in the structure, two of which are involved in 
the internal water bridge referred to earlier. In the third 
0-H S O  hydrogen bond, the water molecule accepts 
a proton from the terminal carboxyl group of an 
adjacent peptide molecule. 
Fig. 2. Crystal structure as viewed along the b axis. The interchd;n 
N-H - - ' 0 hydrogen bonds and the hydrogen bonds involving 
the water molecule are indicated by broken lines and dotted lines 
respectively. Only those atoms involved in hydrogen bonds are 
numbered. 
Table 4. Hydrogen-bond parameters 
The standard deviations are given in parentheses. 
D A  D . .  . A  H-D.. . A  
0(35).*.0(10)" 2.765 (8) A 25 (6)' 
13 ( 5 )  N(11)..*0'(26)* 2.900 (8) 
14 (4) N( 16). * *Of(  I5)c 2.944 (8) 
O( 3 5) * * O( 34)" 2.761 (8) 15 (6) 
0(33)...Of(35)c 2.578 (9) 10 (6) 
Symmetry code: (a) x,y,z; (b) 4 + x, f - y, -z; (c) t + x - 1,4  - y, 
-Z. 
Another interesting feature of the crystal structure is 
the spatial segregation of non-polar and polar groups. 
In fact, they form alternating layers perpendicular to 
the c axis, one layer made up of the phenyl and the 
pyrrolidine rings and the other consisting largely of 
peptide units and water molecules. 
The authors thank the University Grants Com- 
mission, India, and the Indian National Science 
Academy for financial assistance. 
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Internal water bridge and antiparallel sheet in the structure of benzyloxycarbonyl-L-alanyl-D-phenylalanyl-L-proline monohydrate

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Internal water bridge and antiparallel sheet in the structure of benzyloxycarbonyl-L-alanyl-D-phenylalanyl-L-proline monohydrate

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