Protein folding prolyl isomerization

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. 

Tertiary amides, such as those associated with prolyl amide bonds frequently influence turn architectures. The importance of the cis Xaa-Pro bond on activity was recognized and proposed to be the source of differentiation in biological activity [86] therefore, isomerization of the prolyl amide bond is central to regulation of protein folding, immunosuppression, and mitosis. These functions are not surprisingly associated with several disease states and thus substitution of the acyl-proline amide bond with the fluoroolefin isostere has received considerable attention. 

Chyraotrypsin inhibitor 2 (CI2) folds rapidly by simple two-state kinetics that is, D N, with a r1/2of 13 ms.18,19 CI2 is a small 64-residue protein that has all its peptidyl-proline bonds in the favorable trans conformation.20 (There are, of course, additional slow cis —> trans peptidyl-prolyl isomerization events, which account for about 20-30% of the refolding amplitudes.) The occurrence of two-state kinetics does not prove that there are no intermediates on the folding pathway there could be intermediates that are present at high energy and are kineti-cally undetectable (see section B4). Two-state behavior has subsequently been found for many other small proteins. The simplicity of two-state folding kinetics provides the ideal starting point for the analysis and illumination of the basic principles of folding. 

Syn-anti isomerization N-terminal to prolyl peptide fragments is one of the rate limiting steps in protein folding [1], Prolyl fragments exist essentially either in the syn or anti form in native proteins, but as an equilibrium... 

The importance of prolyl peptide bond isomerizations for protein folding is indicated by the following experimental observations. 

Most of the small proteins that were used initially as substrates to test the function of prolyl isomerases contained disulfide bonds, which were left intact during unfolding and refolding. These proteins were used because their unfolding is reversible under a wide variety of conditions and because good evidence existed for a number of them that prolyl isomerizations were involved as rate-limiting steps in their slow-folding reactions. A protein chain without disulfides should be a better model... 

The characterization of the molecular nature of rate-limiting steps is a major aim in the elucidation of the folding mechanism of proteins. It is now clear that cis—trans isomerizations of prolyl peptide bonds can be such slow steps. Folding reactions that involve prolyl isomerization were traditionally identified by measuring their kinetic properties and by comparing them with the properties of prolyl isomerization in short peptides (Brandts et ai, 1975 Nall et al., 1978 Schmid and Baldwin, 1978). 

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