Peptidyl proline

Peptidyl hydroxyprohne and hydroxylysine are formed by hydroxylation of peptidyl proline or lysine in reactions catalyzed by mixed-function oxidases that require vitamin C as cofactor. The nutritional disease scurvy reflects impaired hydroxylation due to a deficiency of vitamin C. 

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... 

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. 

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). 

If, for example, there is a peptidyl-proline bond in the denatured state that has an equilibrium constant ATiso (= [DdJ/[DfranJ), then the equilibrium constant for unfolding will be increased by a factor of 1 + fiso, since [DdJ] + [D,ra J = (1 + iSo)[Dfran.j] Since Kiso is generally in the range of 0.05 to 0.2, this is a small factor if there are just three or four trans-peptidyl prolines in the native state. But if N contains a ds-peptidyl-proline bond, then this leads to very large factors (5 to 20) for each such prolyl residue. 

Proline is an exceptional amino acid residue in that the cis-trans equilibrium only slightly favors the trans form in peptidyl-proline bonds. Small proline-containing peptides in solution contain some 5 to 30% of the cis (syn) isomer, as opposed to less than 0.1% of the cis isomers of the other amino acids.14 The cis form is even found in native proteins two of the four prolines in ribonuclease A... 

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. 

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). 

In the synthesis of l,2,5-triazepine-l,5-diones, which are expected to mimic the structural features of or-peptidyl prolin-amides, the preparation of N2,N3-disubstituted derivatives 213a from the reaction of (Z)-alanine with the N2-substituted triazepines 213 resulted in lower yields. It has been reported that these fused triazepinediones could be elaborated to give constrained rir-peptidyl proline peptide mimetics of defined stereochemistry and sequence <1997J(P1)2297>. 

Collagen, a family of fibrous proteins, is produced by a variety of cell types but principally by fibroblasts (cells found in interstitial connective tissue), muscle cells, and epithelial cells. Type I collagen [collagen(l)], the most abundant protein in mammals, is a fibrous protein that is the major component of connective tissue. It is found in the extracellular matrix (ECM) of loose connective tissue, bone, tendons, skin, blood vessels, and the cornea of the eye. Collagen(l) contains approximately 33% glycine and 21% proline and hydroxyproline. Hydroxyproline is an amino acid produced by posttranslational modification of peptidyl proline residues (see Chapter 7, section V.C., for an earlier introduction to collagen). 

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. ... 

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). 

Peptidyl proline-4-hydroxylase (E.C. 1.14.11.2 - (L)-Prolyl-glycyl peptide, 2-oxoglutarate oxygen oxidoreductase (4-(i )-hydroxylat-ing) (2, 24J). 

Hydrox5q)roline (5) is a major constituent of the cell walls of plants. This compound is generated by posttransla-tional hydroxylation of peptidyl proline both plants and animals require diatomic oxygen for this reaction (Prockop et al., 1962). /ra j-4-Hydroxyproline accounts for as much as 20% of the protein amino acids of primary cell walls. 

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