CHAIN-LIKE CONFORMATION OF HEAT-DENATURED RIBONUCLEASE-A AND CYTOCHROME-C AS EVIDENCED BY SOLUTION X-RAY-SCATTERING

Background: Although the characterization of heat-denatured proteins i s essential for understanding the thermodynamic mechanism of protein f olding, their structural features are still unclear and controversial. In order to address this problem, we studied the size and shape of th e heat-denatured states of bovine ribonuclease A (RNase A) and horse f erricytochrome c (cytochrome c) by solution X-ray scattering, Results: RNase A has four disulfide bonds, whereas cytochrome c, with a covale ntly bound heme group, has no disulfide bond. Guinier plots show that the heat-denatured RNase A is relatively compact, but the heat-denatur ed cytochrome c is expanded. On the other hand, the Kratky plots of th e two proteins are similar, indicating that the heat-denatured protein s assume a chain-like disordered conformation. The X-ray scattering of RNase A and cytochrome c at various temperatures confirmed that their thermal transitions from a globular native state to a chain-like exte nded conformation can be approximated well by a two-state transition. Conclusions: These results indicate that the heat-denatured RNase A an d cytochrome c are substantially unfolded according to the criteria of solution X-ray scattering, although the heat-denatured RNase A remain s compact because of the presence of the disulfide bonds. The results also confirm that the thermal denaturation occurs cooperatively with t he breakdown of secondary and tertiary structure. (C) Current Biology Ltd ISSN 1359-0278.