Prolyl bonds

For most collagens, the folding of the triple helical domain proceeds from the carboxyl end toward the amino end of the trimeric molecule in a zipper-like fashion with a rate that is limited by cis—trans isomerization of peptidyl prolyl bonds." The fast propagation of the triple helix formation is followed by a slower folding... 

This endopeptidase [EC 3.4.24.16] catalyzes the hydrolysis of peptide bonds, preferentially cleaving neurotensin between Pro-10 and Tyr-11. However, there is no absolute requirement for a prolyl bond. 

This manganese-dependent enzyme [EC 3.4.11.5] catalyzes the release of an N-terminal prolyl residue from a peptide. The mammalian enzyme, which is not specific for prolyl bonds, is possibly identical with cytosol amino-peptidase [EC 3.4.11.1]. 

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... 

Prolyl isomerases of the cyclophilin type show some properties that would be expected for a catalyst of cellular protein folding. Cyclophilins occur in all cellular compartments where folding reactions occur. The activity toward accessible prolyl bonds is high, and the specificity with regard to the chemical nature of residue Xaa is low. Additional experiments are clearly needed, however, to clarify the possible role of prolyl isomerases for the in vivo folding process of nascent proteins. 

Landon, M. (1977) Cleavage at aspartyl-prolyl bonds. Methods Enzymol. 47, 145-149. 

Landon, M., Cleavage at aspartyl-prolyl bonds, Meth. EnzymoL, 47, 145-149, 1977. Bomstein, P. and Balian, G., Cleavage at Asn-Gly bonds with hydroxylamine, Meth. EnzymoL, 47, 132-145, 1977. 

Clearly, CTI leads to a periodic backbone contraction/expansion of the polypeptide chain involved, as could be inferred from the isomer-specific distances of the Ca atoms directly attached to the isomerizing peptide bond. For prolyl bonds in native proteins this distance is about 0.8 A shorter in the cis isomer when compared to the respective trans isomer [12]. This atomic translation produces a mechanical moment that was hypothesized to be involved in the functional cycle of motor proteins [13]. 

Interaction of peptides with Li+ ions in dry solvents, such as trifluorethanol (TFE) and tetrahydrofurane, can dramatically influence the free energy difference that discriminates the cis from the trans isomers for both prolyl bonds and secondary amide peptide bonds [56-59]. However, the Li+-induced increase of the cis isomer population in linear oligopeptides depends on the nature of the amino acids preceding proline (G. Fischer, unpublished results). 

Posttranslational modifications considerably extend the chemical diversity of the building blocks forming polypeptide chains. A good deal of effort has been directed toward understanding how the individual amino acid derivative affects the CTI of neighboring prolyl bonds in peptides and proteins. 

Recently, much effort has been put into predicting the isomeric state of prolyl bonds in proteins [129,130], A program on the basis of a secondary structure information was developed that predicts for a given sequence whether a particular peptide bond is in either cis or trans conformation [131]. 

By use of site-directed mutagenesis in positions covering cis prolyl bonds, the proline has been replaced by nonproline amino acids. It came as a surprise that the secondary amide peptide bond formed in the substitution still adopts the thermodynamically disfavored cis conformation in many cases [25,26,132-135], Thus, to overcome the free energy costs of a cis secondary amide peptide bond of about 15 kj mol-1 the structural consequences favoring the trans conformation must be absent in the folded protein variant. Consequently, the CTI is largely retained in these protein variants [133],... 

The X-ray crystal structure database led us to believe that peptide bonds adopt either the cis or trans conformation in native proteins [22,128]. However, NMR spectroscopy [143], and in a few cases, crystal structure analysis [144], provide encouraging experimental evidence of conformational peptide bond polymorphism of folded proteins. Furthermore, conformational changes in response to ligand binding, crystallization conditions and point mutations at remote sites are frequent. Consequently, the three-dimensional protein structure database contains homologous proteins that have different native conformations for a critical prolyl bond [12]. 

For instance, the putative involvement of Cypl8 in the elimination of damaged and misfolded proteins produced by oxidative stress from cells has been found when exploring the cause of familial amyotrophic lateral sclerosis [67]. Cypl8 acts catalytically on still unknown prolyl bonds either by direct participation in protein degradation or indirectly by supporting the functional structure of a Cu/Zn superoxide dismutase-1. A mutant protein of the latter enzyme is responsible for the... 

The dynamics of CTI demonstrate the fundamental similarity between prolyl bonds and secondary amide peptide bonds, making it probable that enzymes exist for the rate acceleration of both types of reactions. The relatively low spontaneous rates indicate the potential importance of CTI of secondary amide peptide bonds as rate-limiting step in protein backbone rearrangements preceding the formation of biologically active proteins. 

Fig. 10.4 Resonance structures of the prolyl bond and potential transition state structures in the catalytic pathway of PPIases. Noncova-lent stabilization of a twisted carbonyl-proline moiety (A). Electrostatic stabilization of the...

Fig. 10.7 Proposed mechanism for enzymatic catalysis of prolyl cis-trans isomerization using two-dimensional representation of the reactant structures for Cypl 8. Only those Cypl 8 residues whose mutagenesis was highly critical to enzyme activity are shown. Arrows symbolize electron redistribution during approaching the transition state. As the prolyl bond rotates and the carbonyl carbon atom develops a positive charge in the transition state, the weak interaction of the base B with the amide proton of Gln63 becomes strong.

In folded proteins the peptide bonds are usually in the trans conformation, which, for nonprolyl bonds,1 is much less strained than the energetically unfavorable cis conformation. For the peptide bonds that precede proline (prolyl bonds), however, the energy difference between the cis and trans states is small, and therefore cis prolyl bonds are found rather frequently in folded proteins. These cis prolyl bonds create a problem for protein folding. The incorrect trans forms predominate in the unfolded or nascent protein molecules, and the trans —> cis isomer-izations are intrinsically slow reactions because rotation about a partial double bond is required. Incorrect prolyl isomers in a protein chain strongly decelerate its folding. This is clearly seen for small single-domain proteins. Many of them refold within a few milliseconds when they contain correct prolyl isomers but when incorrect isomers are present, folding usually requires seconds to minutes. 

JTo facilitate reading I use the terms cis and trans proline for proline residues preceded by a cis or a trans peptide bond in the folded protein nativelike and incorrect, nonnative denote whether or not a particular prolyl peptide bond in an unfolded state shows the same conformation as in the native state. Further, I use the expression isomerization of Xaa for the isomerization of the peptide bond preceding Xaa. Peptide bonds preceding proline are referred to as prolyl bonds, and those preceding residues other than proline are termed as nonprolyl bonds. The folding reactions that involve Xaa—Pro isomerizations as rate-limiting steps are called proline-limited reactions. 

The peptide bonds that precede proline (prolyl bonds) (Fig. 1) are much more often found in the cis conformation because here the cis and trans conformations differ only slightly in energy. In short unstructured peptides cis contents of 10-30% are frequently observed (Cheng and Bovey, 1977 Grathwohl and Wuthrich, 1981 Reimer et al., 1998). The actual cis/trans ratio depends on the size and chemical nature of the flanking amino acids. 

In folded proteins the conformational state of a prolyl bond is usually well defined, because in most cases only one of the two conformations (cis or trans) can be accommodated in the folded structure. Of 1435 nonredundant protein structures in the Brookhaven protein database, 43% contain at least one cis peptidyl-prolyl bond (Reimer et al., 1998), and 7% of all prolyl peptide bonds in folded proteins are cis (Stewart et al., 1990 Macarthur and Thornton, 1991). 

A mixture of species is thus created, which consists of a single unfolded form with correct prolyl isomers (Uf) and one or more unfolded species with incorrect prolyl isomers (Ug). The Up molecules with the nativelike prolyl isomers refold directly (and often quickly) to the native conformation. Ug molecules, however, refold slowly, because refolding is coupled with the reisomerizations of the incorrect prolyl bonds. 

Cis/trans isomerism is not confined to prolyl bonds. Cis peptide bonds to residues other than proline (cis nonprolyl bonds) are, however, extremely rare in folded proteins because the trans form is strongly favored over cis. In short unstructured peptides 99.5—99.9% of nonprolyl peptide bonds are in the trans state (Scherer et al., 1998). Proteins that contain nonprolyl cis peptide bonds in their native states must therefore undergo trans —cis isomerizations of these bonds in virtually all refolding molecules. 

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