| dc.description.abstract | Unlike polyproline, which undergoes mutarotation between the right-handed helical form I, with all peptide bonds in the cis orientation, and the left-handed helical form II, which has all trans peptide bonds, polyhydroxyproline does not mutarotate between two ordered structures in solution. The ordered structure of PHP in solution is not stabilized by any intermolecular interactions, such as those found for PHP in form A in the solid state. The structure is also not stabilized by any firmly bound water molecules, since IR studies in D?O indicate that the water molecules are easily removable and that rapid exchange occurs between bound and free D?O molecules at high humidity. CD studies indicate that PHP does not undergo any structural transformation when cast as a film from an aqueous solution. Our X-ray studies have shown that PHP films, when prepared by slow evaporation from aqueous solution, are in the intrachain hydrogen-bonded form B. Since at high humidity (>66% RH) it is always the B-form X-ray pattern which is observed, we conclude from our studies that the ordered structure of PHP in solution is also the form B and not form A.
Several previous studies also support our conclusion. It was found by Bensing and Pysh (1971) that PHP films, when cast from aqueous solution in the presence of an electric field, show parallel dichroism at 218 and 188 nm in the ultraviolet absorption spectra. Since the observed spectrum differs from that observed for PPII, these workers suggested that PHP has probably undergone mutarotation to a form with cis peptide bonds. However, there are also considerable differences in the absorption bands observed for PPI and the PHP film. Since in the structure proposed by us for the form B of PHP, the carbonyl groups are oriented relatively parallel to the helix axis (Bansal et al., 1978), it is quite likely that it was the B form which was observed by these workers.
It is worth mentioning here that the hydrodynamic properties of polyhydroxyproline as well as polyproline apparently exhibit molecular weight dependence. The low molecular weight samples are found to have the conformation of a somewhat flexible rod, while the high molecular weight samples behave hydrodynamically like a random coil (Clark and Mattice, 1977). However, as pointed out by Tanaka and Scheraga (1975), the hydrodynamic behaviour of these polypeptides is ill-understood. The available CD data, including our own, do not show any apparent molecular weight dependence over a range from 9,000 to 27,000. The ordered conformation of PHP that contributes to this CD is, according to us, the form B, both in solution and in the film.
Solution studies of PP and PHP using proton NMR (Torchia, 1971, 1972), 13C NMR and Raman spectroscopy (Torchia and Lyerla, 1974; Deveney et al., 1971) also indicate that there are some marked differences in the structures of the two polymers in solution. In particular, while the pyrrolidine ring is quite flexible in PPII, it is relatively rigid in PHP, indicating that the interaction of the ?-hydroxyl group with the backbone carbonyl groups renders the ring immobile to some extent. These studies, as well as the observations that the PHP helix is considerably more stable than the PPII helix in aqueous solution (Mattice and Mandelkern, 1970), support our conclusion that the ordered structure of PHP in solution is the form B, with the ?-hydroxyl group forming an intrachain hydrogen bond with a backbone carbonyl oxygen. The possibility of such hydrogen bond formation was suggested earlier for the polymer (Gly-Hyp)n by Mattice and Mandelkern (1971), while Torchia (1972), from proton NMR studies, had proposed a similar scheme for PHP.
From theoretical considerations, it has been shown in Section 6.3.1 that, though the presence of the ?-hydroxyl group does not put any stereochemical restriction on the PHP chain taking up a structure with all cis units, no intrachain hydrogen bonds, either direct or water-bridged, are possible for this structure. Hence, the absence of this structure may be due to the fact that, in solution, once three consecutive units take up trans orientations, a hydrogen bond is formed linking the ?-hydroxyl group of the (i+1)th residue to the carbonyl oxygen of the (i?1)th residue, and the polypeptide chain is locked in this thermodynamically favourable conformation. Thus, the occurrence of the intrachain hydrogen-bonded B-form structure readily explains the absence of mutarotation and the great stability of PHP in solution.
The implication of this type of intramolecular stabilization through the ?-hydroxyl group, on the specific hydroxylation of proline residues in collagen, will be discussed in Chapter 8 in detail. | |
