Ribonuclease refolding

Recently, SPE was determined to be useful in a protein refolding process. Goto, Hatton, and coworkers demonstrated that SPE was the most efficient method for solubilizing denatured ribonuclease into AOT-based w/o-MEs [59]. The w/o-ME encapsulated... 

J.-R. Garel and R. L. Baldwin, Both the fast and slow refolding reactions of ribonuclease A yield native enzyme, Proc. Natl. Acad. Sci. U.S.A. 70, 3347-3351 (1973). 

A. Rehage and F. X. Schmid, Fast- and slow-refolding forms of unfolded ribonuclease A differ in tyrosine fluorescence, Biochemistry 21, 1499-1505 (1982). 

F. X. Schmid, R. Grafl, A. Wrba, and J. J. Beintema, Role of proline peptide bond isomerization in unfolding and refolding of ribonuclease, Proc. Natl. Acad Sci. U.S.A. 83, 872-876 (1986). 

The smallest member of a new family of prolyl iso-merases (unrelated to the cyclophilins or the FK-506 binding proteins) that catalyzes the proline-limited folding of a variant of ribonuclease T1 with a KJK value of 30,000 M s With the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide as a substrate in parvulin-catalyzed prolyl isomerization, this parameter is 1.1 x 10 M s Parvulin also accelerates its own refolding in an autocatalytic fashion. 

G3. Garel, A. T., and Garel, J. R., Partially oxidized active intermediates in refolding of reduced ribonuclease. J. Biol. Chem. 257, 1031 1033 (1982). 

Enzymatic activity, however, is not merely associated with covalent structures, but chiefly with tertiary structure which is still more difficult to determine. The crucial role of tertiary structure is proved by the fact that denaturation brings about inactivation. Even with proteins which may be reversibly denatured, such as chymotrypsin and trypsin, activity is lost as long as denaturation persists. Ribonuclease appeared for a while to be an exception, since it was still active in 8 M urea. But it was shown later that phosphate ions, at a concentration as low as 0.003 M, and polyphosphates induced in urea-denatured ribonuclease spectral changes usually associated with refolding (164). It could then be assumed that ribonucleic acid, the actual substrate, was also able to refold the denatured form and prevent inactivation in this way. In other words, even in ribonuclease, the active center is probably not built by adjacent residues in a tail or a ring, but by some residues correctly located in space by the superimposed... 

Panick, G., Winter, R. Pressure-induced unfolding/refolding of ribonuclease A static and kinetic Fourier transform infrared spectroscopy study, Biochem. 39 (2000) 1862-1869. 

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