Solvent Perturbation and Ultraviolet Optical Rotatory Dispersion Studies of Paramyosin

The location of the tyrosyl residues (there are no tryptophyl residues) in paramyosin was studied by solvent perturbation difference spectroscopy, with 20% polyhydroxy perturbants of increasing size from ethylene glycol to sucrose. Of the tyrosyl residues, 35% are not perturbed by any of the solvents, and an additional 10 to 15% apparently lie in crevices which are inaccessible to perturbants with diameters greater than 5 A (i.e. glycerol and larger molecules). These latter groups may be involved in the intermolecular aggregation phenomenon. Increasing concentrations of guanidine-HCl increase the magnitude of the denaturation blue shift in four steps. The first two of these steps coincide with the first two steps in the helix-coil transition and account for the exposure of about 68% of the 38 tyrosyl groups to the solvent. The remaining 12 tyrosyls are exposed in two steps during the breakdown of the final third of the helix and are considered to be the same 12 groups inaccessible to all perturbants. The ultraviolet optical rotatory properties of native α-helical paramyosin from 315 to 190 mµ were studied in detail. Statistical analyses of the Moffitt equation showed that it adequately describes the dispersion data in the 315 to 240 mµ region, if λ0 is taken as 220 mµ, until the molecule becomes less than 30% helical. Beyond that point the one-term Drude equation is sufficient. The Moffitt parameters are shown to be colinear with [m']232 for the whole helix-coil transition. By comparison with optical rotatory dispersion parameters for random polypeptides, paramyosin retains approximately 10% order in 7 m guanidine. Since this order cannot be destroyed by heating to 50° or by 8 m guanidine-HCl and the molecule refolds completely upon removal of the guanidine, hydrophobic interactions appear responsible for this order.