STEPS IN THE PATHWAY OF THE THERMAL UNFOLDING OF RIBONUCLEASE-A - NONSPECIFIC PHOTO-CHEMICAL SURFACE-LABELING STUDY

The technique of nonspecific photochemical surface labeling is used to study the thermal unfolding of ribonuclease A at pH 5. The results indicate that the unfolding process consists of a minimum of three overlapping stages. Below 35 °C, there are localized changes which involve Met-13 as well as some Tyr and Ala residues. From other work on the ultraviolet absorption spectrum and proteolytic susceptibility of ribonuclease A, the latter residues are tentatively identified as Tyr-92 and Ala-19 and/or Ala-20. Between 35 and 40 °C, the outer shell of the ribonuclease structure which is most exposed to the solvent begins to unfold in a broad transition that is not complete until 70 °C. This stage overlaps the partial unfolding of a nonpolar core which is composed of Phe, Val, Leu and lie residues. This core unfolds between 60 and 70 °C. The unfolded form of ribonuclease A at 78 °C and pH 5 still has some residual structure. The observation that the nonpolar core of ribonuclease A becomes exposed to the solvent only incompletely and at a late stage of the unfolding process suggests that the nonpolar residues are important in the initial stage (nucleation) of protein folding. This is consistent with the concept that hydrophobic interactions dominate in the nucleation process [Matheson, R. R., & Scheraga, H. A., 1978), Macromolecules 11, 819]. The advantages and disadvantages of the nonspecific photochemical surface labeling technique as one for studying protein unfolding are examined. © 1979, American Chemical Society. All rights reserved.