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  • Title: Structure of a hydrophobically collapsed intermediate on the conformational folding pathway of ribonuclease A probed by hydrogen-deuterium exchange.
    Author: Houry WA, Scheraga HA.
    Journal: Biochemistry; 1996 Sep 10; 35(36):11734-46. PubMed ID: 8794754.
    Abstract:
    The unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species which have different X-Pro peptide bond conformations. One of these unfolded species, labeled Uvf, has all its X-Pro peptide bonds in the native conformation. Therefore, the refolding of Uvf is a purely conformational folding process which is not complicated by cis-trans X-Pro peptide bond isomerization. There are two identifiable intermediates on the folding pathway of Uvf: one which is a largely unfolded intermediate (IU) and another which is a hydrophobically collapsed intermediate (I phi). An instrument was built, and experiments were designed to study the structure in IU and I phi by hydrogen-deuterium exchange. These experiments are a combination of a double-jump experiment followed by a pulse-labeling experiment. The native protein was first unfolded to populate Uvf to more than 99%, and then Uvf was refolded for a specified period of time. After refolding, hydrogen-deuterium exchange of the backbone amides was initiated for a given time by raising the pH. Subsequently, the exchange was quenched and the protein was allowed to continue to fold to the native state. The extent of exchange was determined quantitatively by two-dimensional NMR spectroscopy. The data indicate that IU has no secondary structure that can protect the backbone amides from exchange under the conditions employed. On the other hand, in I phi, the second helix (residues 24-34) and a large part of the beta-sheet region of the protein are formed, while the rest of the protein molecule remains unstructured. In general, the protection factors in I phi are low, indicating that this intermediate has a dynamic structure. Our observations are consistent with I phi being a molten-globule-like intermediate. The regular structure formed in I phi is much less than that observed in a hydrogen-bonded intermediate (Ii) populated early on the major slow-refolding pathway of the protein [Udgaonkar, J. B., & Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197-8201]; in addition, the structure in I phi has much lower stability than that in Ii. This implies that a slower refolding rate allows for a higher cooperativity between the different structural elements of the protein, resulting in the formation of more stable (native-like) intermediates (as in Ii) during the folding process.
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