Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow.
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-denatured 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 (less than 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 of a new structure(s) that lie(s) outside of 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.
Adler
M
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Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods; D010455:Peptides; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D010455:Peptides; D012259:Ribonuclease, Pancreatic