Peptidyl proline isomerization

That peptidyl-proline isomerization is a post-unfolding event is readily demonstrated by double-jump experiments.17 The protein is denatured for several milliseconds and then restored to renaturing conditions before the peptidyl prolines have had time to isomerize. The proline-related phases are then no longer seen. 

CI2 was used in the last chapter to exemplify the basic kinetics of two-state reactions. The small fraction that folds slowly because of cis —> trans peptidyl-proline isomerization is ignored in the following discussion. CI2 folds according to first-order kinetics from a relatively open denatured state, with a half-life of some 10 ms.25 Some mutants fold 10 times faster. 

The early stages of folding of barstar have been measured on the microsecond time scale by temperature jumping its cold-denatured state from 2 to 10°C.65,66 There is the fast formation of a folding intermediate (tm 200 fxs) with the peptidy 1-proline 48 bond trans, followed by the formation with ty2 60 ms of a second intermediate that is highly native-like because it binds to and inhibits barnase. The native-like intermediate then undergoes trans cis peptidyl-proline isomerization on the time scale of minutes to give the final native structure (equation 19.2). 

A correct folding of recombinant proteins is regulated by the redox potential of the outer compartments and performed by foldases. These catalyse disulfide bond formation and peptidyl-proline isomerization, prevent protein a regation in inclusion bodies and hamper the proteolysis by cytoplasmic enzymes. The coexptession of eukaryotic foldases leads to an increased yield of eukaryotic recombinant proteins in E. colif Moreover, the supply of reduced glutathione generates favourable environmental conditions for carrying out a correct folding in the periplasm. ... 

Two structurally unrelated immunosuppressant drugs, cyclosporin A and FK506, have been shown to bind to separate proteins, which have in common the ability to catalyse the interconversion (8) of the cis and trans rotamers of peptidyl-proline bonds of peptide substrates. A profound change in the conformation, and hence the shape and binding properties of the protein, may result. The mechanism of this isomerization appears, on the basis of recent work (Rosen et al., 1990 Van Duyne et al., 1993 Albers et al., 1990), to involve simple twisting about the amide bond, rather than such alternatives as conversion to a C-N single bond by addition of a nucleophile to C=0.y The proteins which catalyse the reaction may be... 

Equilibrium parameters derived from kinetics will differ from those measured at equilibrium because of the effects of proline isomerization. The additional equilibria between cis- and rans-peptidyl-proline bonds in the denatured state add to its stability and so favor denaturation (equation 18.1). 

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. 

Shift the reaction path (cyclophilin). Cyclophilin, which catalyzes the cis-trans isomerization of peptidyl-proline bonds, is converted into protease by an engineered introduction of the proteolytic triad, Asp-His-Ser (Qu6m6neur et al, 1998). 

Disulfide bond formation within the individual propeptides precedes folding and trimers are then formed by association of the C-terminal propeptides." Disulfide bonds between the chains are then formed and this formation is most likely catalyzed by PDI." As triple helix formation proceeds, the rate-limiting step in this process is the cis—trans isomerization of peptidyl-Pro bonds. This process can be catalyzed by peptidyl-prolyl cis—trans isomerases (cyclophilins and FKBPs). This activity is required to convert the proline residues to the trans form required for triple helix formation." " " ... 

Refolding is generally found to proceed by a series of exponential phases. Many of these exponentials are a consequence of cis-trans isomerization about peptidyl-prolyl bonds.14,15 The equilibrium constant for the normal peptide bond in proteins favors the trans conformation by a factor of 103-104 or so. The peptidyl-prolyl bond is an exception that has some 2-20% of cis isomer in model peptides (see Chapter 1, Figure 1.3). Further, it is often found as the cis isomer in native structures. (Replacement of ds-prolines with other amino acids by protein engineering can retain the cis stereochemistry.16) The interconversion of cis to trans in solution is quite slow, having half-lives of 10-100 s at room temperature and neutral pH. This has two important consequences. First, a protein that has several... 

The peptidyl-prolyl cis/trans isomerase Pin 1 isomerizes the peptide bond of a phos-phorylated serine or phosphorylated threonine followed by a proline. Through isomerization of pSer-Pro or PThr-Pro, Pinl regulates a number of proteins. Together with its ability to regulate phosphorylation and conformation of tau proteins. Pin 1 is considered a potential neuroprotective function against AD. 

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