Dynamic light scattering and circular dichroism experiments were performed to determine the compactness and residual secondary structure of reduced and by 6 M guanidine hydrochloride denatured ribonuclease A. We find that reduction of the four disulphide bonds by dithiothreitol at 20°C leads to total unfolding and that a temperature increase has no further effect on the dimension. The Stokes' radius of ribonuclease A at 20°C is R S = (1.90 4" 0.04) nm (native) and Rs = (3.14 4" 0.06) nm (reduced-denatured). Furthermore, circular dichroism spectra do not indicate any residual secondary structure. We suggest that reduced-denatured Ribonuclease A has a random coil-like conformation and is not in a compact denatured state.
Over a certain time range the results are free of finite-size effects and thus represent correlation functions of an infinite chain (bulk regime) or a semi-infinite chain (boundary regime). In the bulk regime, the long-time asymptotic decay as inferred by extrapolation is Gaussian at T = oo, exponential at 0 < T < oo, and power-law (~ t-1 1 2 ) at T = 0, in agreement with exact results. In the boundary regime, a power-law decay is obtained at all temperatures; the characteristic exponent is universal at T = 0 (~ r 1 ) and at 0 < T < oo (~ r 3 1 2 ), but is site dependent at T = oo. In the high-temperature regime (T I J ~ 1) and in the low-temperature regime (T I J « 1 ), crossovers between different decay laws can be observed in (Sf(t)Sj). Additional crossovers are found between bulk-type and boundary-type decay for i = j near the boundary, and between spacelike and timelike behavior for i -:f. j.
During folding of RNase A an initial global hydrophobicity is not observed, which contradicts the view that this is a general requirement for protein folding. This folding behavior could be typical of similar, moderately hydrophobic proteins.
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