ACID DENATURED APO-CYTOCHROME-C IS A RANDOM COIL - EVIDENCE FROM SMALL-ANGLE X-RAY-SCATTERING AND DYNAMIC LIGHT-SCATTERING

The conformation of a denatured protein has been investigated, since the experimental data on the structure of denatured proteins have been incomplete until now. The Stokes' radius R(s) and the radius of gyration R(g) of apo-cytochrome c at pH 2.3 have been determined by dynamic light scattering and small-angle X-ray scattering, respectively. The values of these structure parameters, extrapolated to zero protein concentration, are R(s) = 3.0 nm and R(g) = 4.6 nm. The ratio R(g) . R(s)-1 is a sensitive indicator of the molecular conformation. The ratio of 1.55 obtained by us is typical for a random-coil polymer. The persistence length - the characteristic of the molecular flexibility - was determined to be a = 1.81 nm. From this results the root-mean-square average end-to-end distance of the molecules <h2>1/2 = 11.2 nm and the characteristic ratio <h2>/n(p)l(p)2 = 8.43, where n(p) = 104 is the number of amino acid residues and l(p) the distance between C-alpha-atoms. We obtained a second virial coefficient A2 = 8.2 . 10(-3) mol cm3 g-2. The experimentally determined structure parameters are in approximate agreement with those predicted by Flory and others for an unperturbed, randomly coiled polypeptide. The expansion factor lies between 1.1 and 1.2. In conclusion, we have shown that apo-cytochrome c at pH 2.3 and at low concentration has the conformation of a perturbed random coil with repulsive potentials between the chain segments.