VOL. 15, 503-506 (1976)
BIOPOLYMERS
An X-Ray Study of
Poly(L-prolyl-L-a-phenylglycyl-L-proline) A. DEL PRA and M. PALUMBO, Institute of Organic Chemistry, Biopolymer Research Center of C.N.R., University of Padova, 35100 Padua, Italy; and M. GOODMAN, Department of Chemistry, University of California, S a n Diego, La Jolla, California 92093
Synopsis X-ray diffraction studies have been made on the polytripeptide poly(L-prolyl-L-a-phenylglycyl-L-proline). Its structure has been found to be helical, with a poly(L-proline) I1 conformation, packed in an orthorhombic lattice, space group P21212, with a = 14.3 A, b = 13.5 A, and c = 9.4 A.
INTRODUCTION Among the characteristic properties of all collagens are the occurrence of glycyl residues in every third position along the length of the protein chains and relatively high content (about 20%) of proline and hydroxyproline residues1 Indeed, these features helped to show that collagen has a triple-helical s t r ~ c t u r e . ~ In - ~ the past few years a number of polytripeptides and polyhexapeptides have been synthesized and studied in order to understand better the details of such a triple-helical structure and the factors that stabilize it. Several of these polymers have been found to have collagenlike structures in the solid state and in ~ o l u t i o n .All ~ such collagen models studied contain glycine a t every third position and have a t least one imino acid for every three residues. Of course, the stability of the collagen triple-helical structure varies for different polymers and some adopt this conformation only under rather special conditions. Sequential copolypeptides containing glycine that is not located a t every third residue, L-proline and L-hydroxyproline, have been also i n ~ e s t i g a t e d . ~ , ~ Some of them assume in solution ordered structures similar to that of poly(L-proline) 11, [(Pro),II]. In this paper we report an X-ray structural investigation on the sequential copolypeptide poly(L-prolyl-L-a-phenylglycyl-L-proline) (Pro@Gly-Pro), where the glycyl residue, responsible for the conformational features of collagen, is replaced by an L-a-phenylglycyl group. 503
0 1976 by John Wiley & Sons, Inc.
504
DEL PRA, PALUMBO, AND GOODMAN
METHODS A N D MATERIALS Specimens of (Pro-@Gly-Pro), prepared and purified as previously reported,8 were photographed as powders and as films and fibers. Films of (Pro+Gly-Pro), were prepared by stroking out a drying film until it was solid. The solvents used were trifluoroethanol and dimethylformamide. Fibers were also made by spinning the same (Pro-@Gly-Pro), samples from a trifluoroethanol solution into diethyl ether. Both the films and the fibers were then heated to about 100°C in uucuo, for several hours, to promote crystallinity. Their density as determined by flotation in KBr aqueous solution was 1.28 g- ~ m - ~ . The X-ray photographs were taken with a flat camera, with sampleto-photographic film distance d variable from 4 to 10 cm and with a cylindrical camera (5.73 cm in diameter). Tilted techniques were also used, with Ni-filtered CuKa radiation from a normal focus X-ray tube and a 0.6-mm pinhole. We were not able to prepare specimens giving appreciably oriented X-ray photographs.
RESULTS AND DISCUSSION Powder photographs of (Pro-@Gly-Pro), (Fig. 1) which do not show resemblance in spacing and intensity distribution to those of unstretched collagen,2 exhibit diffraction rings up to about 3 A.
Fig. 1. Normal beam X-ray diffraction pattern of [Pro-@Gly-Pro],powder; flat camera
d = 6 cm, CuKa radiation.
POLYTRIPEPTIDES
505
TABLE I X-ray Data for (Pro-$Gly-Pro),
200 020 120 021 220 300 112 212 321 222 003 113 142 521
7.15 6.75 6.10 5.48 4.90 4.76 4.24 3.77 3.59 3.39 3.14 2.98 2.69 2.53
7.15 6.75 6.10 5.55 4.90 4.71 4.20 3.75 3.58 3.38 3.14 2.96 2.70 2.50
vs ms W
mb vs ms mb mb vw vw W
W W W
Orthorhombic unit cell: a = 1 4 . 3 8, b = 13.5 8, c = 9.4 8. Abbreviations used: vs, very strong; ms, medium strong; w, weak; mb, medium broad; vw, very weak.
We have been able to measure 14 spacings do reported in Table I, with their estimated intensities. With these data we tried to define the solid-state conformation and the crystalline packing of (Pro-dGly-Pro)n. First, the presence of the 3.14-A reflection is highly suggestive of the (Pro),II-like c o n f ~ r m a t i o n . ~In fact, several polymers with amino acid sequences closely related to (Pro-6Gly-Pro), have been found to assume a similar conformation.1° On this basis we found all the observed reflections can be indexed according to an orthorhombic unit cell with a = 14.3 A, b = 13.5 A, and c = 9.4 A. Miller’s indices are reported in Table I together with the calculated spacing d,. The calculated density is D, = n-0.30 g - ~ m - ~ where , n is the number of helices crossing the cell. Comparison with the observed density, D o = 1.28 g - ~ m - ~ indicates , that such a cell is crossed by four helices and also suggests that some solvent molecules are still in the crystal. As a consequence of the systematic absences, choosing for instance the P21212 space group, the unit cell would contain two couples of symmetrically equivalent (Pro-4Gly-Pro), helices, with a diameter of approximately 7 A. It is very likely that the deviation of (Pro-dGly-Pro), from hexagonal packing arises from steric requirements of the phenyl rings in the interhelix interactions. It may be that the packing is somewhat analogous to the double-layered sheets proposed for (Gly-Pro-Pro),. l2 Experimental data from oriented samples would allow a detailed structural analysis. Taking into account the possibility of correlating the polypeptide conformation in the solid state and in solution, the present results further support the reported conclusions8 that (Pro-@Gly-Pro), in solution
506
DEL PRA, PALUMBO, AND GOODMAN
is in the (Pro), I1 conformation. A collagenlike triple helix, as in poly(L-prolylglycyl-L-proline),ll can be ruled out on the basis of the experimental data. This is supported by the fact that L-a-phenylglycine replaces glycine at every third residue, thus preventing the polymer from associating for steric reasons.
References 1. Hanning, K. & Nordwig, A. (1967) in Treatise on Collagen, Ramachandran, G . N., Ed., Academic, London, Vol. 1, p. 73. 2. Ramachandran, G. N. & Kartha, G. (1955) Nature 176,593-595. 3. Rich, A. & Crick, F. H. C. (1961) J . Mol. Biol. 3,483-492. 4. Cowan, P. M., McGavin, S. & North, A. C. T. (1955) Nature 176,1062-1064. 5. Traub, W. & Piez, K. A. (1971) Aduan. Protein Chem. 25, 290-325 (and references contained therein). 6. Mattice, W. L. & Mandelkern, L. (1971) Biochemistry 10,19261933. 7. Mattice, W. L. & Mandelkern, L. (1971) J. Amer. Chem. SOC.92,5285-5287. 8. Palumbo, M., Rodin, R. & Goodman, M. (1975) Biochemistry 14,485-491. 9. Sasisekharan, V. (1959) Acta Crystallogr. 12,897-903. 10. Segal, D. M. & Traub, W. (1969) J. Mol. Biol. 43,487-496 (and references contained therein). 11. Traub, W. & Yonath, A. (1966) J. Mol. Biol. 16,404-414. 12. Traub, W. (1969) J . Mol. Biol. 43,479485.
Received July 18,1975 Returned for revision August 13,1975 Accepted October 8,1975
BIOPOLYMERS
An X-Ray Study of
Poly(L-prolyl-L-a-phenylglycyl-L-proline) A. DEL PRA and M. PALUMBO, Institute of Organic Chemistry, Biopolymer Research Center of C.N.R., University of Padova, 35100 Padua, Italy; and M. GOODMAN, Department of Chemistry, University of California, S a n Diego, La Jolla, California 92093
Synopsis X-ray diffraction studies have been made on the polytripeptide poly(L-prolyl-L-a-phenylglycyl-L-proline). Its structure has been found to be helical, with a poly(L-proline) I1 conformation, packed in an orthorhombic lattice, space group P21212, with a = 14.3 A, b = 13.5 A, and c = 9.4 A.
INTRODUCTION Among the characteristic properties of all collagens are the occurrence of glycyl residues in every third position along the length of the protein chains and relatively high content (about 20%) of proline and hydroxyproline residues1 Indeed, these features helped to show that collagen has a triple-helical s t r ~ c t u r e . ~ In - ~ the past few years a number of polytripeptides and polyhexapeptides have been synthesized and studied in order to understand better the details of such a triple-helical structure and the factors that stabilize it. Several of these polymers have been found to have collagenlike structures in the solid state and in ~ o l u t i o n .All ~ such collagen models studied contain glycine a t every third position and have a t least one imino acid for every three residues. Of course, the stability of the collagen triple-helical structure varies for different polymers and some adopt this conformation only under rather special conditions. Sequential copolypeptides containing glycine that is not located a t every third residue, L-proline and L-hydroxyproline, have been also i n ~ e s t i g a t e d . ~ , ~ Some of them assume in solution ordered structures similar to that of poly(L-proline) 11, [(Pro),II]. In this paper we report an X-ray structural investigation on the sequential copolypeptide poly(L-prolyl-L-a-phenylglycyl-L-proline) (Pro@Gly-Pro), where the glycyl residue, responsible for the conformational features of collagen, is replaced by an L-a-phenylglycyl group. 503
0 1976 by John Wiley & Sons, Inc.
504
DEL PRA, PALUMBO, AND GOODMAN
METHODS A N D MATERIALS Specimens of (Pro-@Gly-Pro), prepared and purified as previously reported,8 were photographed as powders and as films and fibers. Films of (Pro+Gly-Pro), were prepared by stroking out a drying film until it was solid. The solvents used were trifluoroethanol and dimethylformamide. Fibers were also made by spinning the same (Pro-@Gly-Pro), samples from a trifluoroethanol solution into diethyl ether. Both the films and the fibers were then heated to about 100°C in uucuo, for several hours, to promote crystallinity. Their density as determined by flotation in KBr aqueous solution was 1.28 g- ~ m - ~ . The X-ray photographs were taken with a flat camera, with sampleto-photographic film distance d variable from 4 to 10 cm and with a cylindrical camera (5.73 cm in diameter). Tilted techniques were also used, with Ni-filtered CuKa radiation from a normal focus X-ray tube and a 0.6-mm pinhole. We were not able to prepare specimens giving appreciably oriented X-ray photographs.
RESULTS AND DISCUSSION Powder photographs of (Pro-@Gly-Pro), (Fig. 1) which do not show resemblance in spacing and intensity distribution to those of unstretched collagen,2 exhibit diffraction rings up to about 3 A.
Fig. 1. Normal beam X-ray diffraction pattern of [Pro-@Gly-Pro],powder; flat camera
d = 6 cm, CuKa radiation.
POLYTRIPEPTIDES
505
TABLE I X-ray Data for (Pro-$Gly-Pro),
200 020 120 021 220 300 112 212 321 222 003 113 142 521
7.15 6.75 6.10 5.48 4.90 4.76 4.24 3.77 3.59 3.39 3.14 2.98 2.69 2.53
7.15 6.75 6.10 5.55 4.90 4.71 4.20 3.75 3.58 3.38 3.14 2.96 2.70 2.50
vs ms W
mb vs ms mb mb vw vw W
W W W
Orthorhombic unit cell: a = 1 4 . 3 8, b = 13.5 8, c = 9.4 8. Abbreviations used: vs, very strong; ms, medium strong; w, weak; mb, medium broad; vw, very weak.
We have been able to measure 14 spacings do reported in Table I, with their estimated intensities. With these data we tried to define the solid-state conformation and the crystalline packing of (Pro-dGly-Pro)n. First, the presence of the 3.14-A reflection is highly suggestive of the (Pro),II-like c o n f ~ r m a t i o n . ~In fact, several polymers with amino acid sequences closely related to (Pro-6Gly-Pro), have been found to assume a similar conformation.1° On this basis we found all the observed reflections can be indexed according to an orthorhombic unit cell with a = 14.3 A, b = 13.5 A, and c = 9.4 A. Miller’s indices are reported in Table I together with the calculated spacing d,. The calculated density is D, = n-0.30 g - ~ m - ~ where , n is the number of helices crossing the cell. Comparison with the observed density, D o = 1.28 g - ~ m - ~ indicates , that such a cell is crossed by four helices and also suggests that some solvent molecules are still in the crystal. As a consequence of the systematic absences, choosing for instance the P21212 space group, the unit cell would contain two couples of symmetrically equivalent (Pro-4Gly-Pro), helices, with a diameter of approximately 7 A. It is very likely that the deviation of (Pro-dGly-Pro), from hexagonal packing arises from steric requirements of the phenyl rings in the interhelix interactions. It may be that the packing is somewhat analogous to the double-layered sheets proposed for (Gly-Pro-Pro),. l2 Experimental data from oriented samples would allow a detailed structural analysis. Taking into account the possibility of correlating the polypeptide conformation in the solid state and in solution, the present results further support the reported conclusions8 that (Pro-@Gly-Pro), in solution
506
DEL PRA, PALUMBO, AND GOODMAN
is in the (Pro), I1 conformation. A collagenlike triple helix, as in poly(L-prolylglycyl-L-proline),ll can be ruled out on the basis of the experimental data. This is supported by the fact that L-a-phenylglycine replaces glycine at every third residue, thus preventing the polymer from associating for steric reasons.
References 1. Hanning, K. & Nordwig, A. (1967) in Treatise on Collagen, Ramachandran, G . N., Ed., Academic, London, Vol. 1, p. 73. 2. Ramachandran, G. N. & Kartha, G. (1955) Nature 176,593-595. 3. Rich, A. & Crick, F. H. C. (1961) J . Mol. Biol. 3,483-492. 4. Cowan, P. M., McGavin, S. & North, A. C. T. (1955) Nature 176,1062-1064. 5. Traub, W. & Piez, K. A. (1971) Aduan. Protein Chem. 25, 290-325 (and references contained therein). 6. Mattice, W. L. & Mandelkern, L. (1971) Biochemistry 10,19261933. 7. Mattice, W. L. & Mandelkern, L. (1971) J. Amer. Chem. SOC.92,5285-5287. 8. Palumbo, M., Rodin, R. & Goodman, M. (1975) Biochemistry 14,485-491. 9. Sasisekharan, V. (1959) Acta Crystallogr. 12,897-903. 10. Segal, D. M. & Traub, W. (1969) J. Mol. Biol. 43,487-496 (and references contained therein). 11. Traub, W. & Yonath, A. (1966) J. Mol. Biol. 16,404-414. 12. Traub, W. (1969) J . Mol. Biol. 43,479485.
Received July 18,1975 Returned for revision August 13,1975 Accepted October 8,1975
