Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow

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Abstract
A new technique, continuous recycled flow (CRF) spectroscopy, has been developed for observing intermediates of any thermally induced, reversible reaction with a half-life of 10 s or longer. The structure can be probed by any spectroscopic method which does not perturb the system. Prolonged signal acquisitions of 8 h for ribonuclease A are possible. CRF was used to investigate the structure of the slow-folding intermediates of chemically intact ribonuclease A (RNase A) during thermal unfolding/folding under acidic conditions. The following conclusions were reached on the basis of the proton nuclear magnetic resonance and far-ultraviolet circular dichroism spectra of a folding intermediate(s): (A) The conformation of the detected folding intermediate(s) is similar to that of the heat-denaturated protein. There is only limited formation of new structures. (B) The N-terminal .alpha.-helix is partially stable under these conditions and is in rapid (< 10 ms) equilibrium with the denatured conformation. (C) There are long-range interactions between the hydrophobic residues of the N-terminal .alpha.-helix and the rest of the protein. These interactions persist well above the melting point. (D) An aliphatic methyl group reports on the formation a new structure(s) that lie(s) outside the N-terminal region. (E) The structures detected in chemically modified nonfolding forms of the RNase A are also present in the folding intermediate(s). There are, however, additional interactions that are unique to chemically intact RNase A.