Cis and trans proline

A large number of 13C NMR studies on proline derivatives and proline peptides have appeared in the literature [815-830]. As the electron charge density of cis-proline carbons is different from that of franx-prolinc carbons, these isomers can be differentiated by nCNMR spectroscopy [826, 830]. On the basis of calculations Tonelli [831] predicted four conformations for the dipeptide Boc-Pro-Pro-OBzl, three of which could be detected by 13C NMR spectroscopy [826, 830], In proline-containing peptides the stereochemistry of the proline residue plays an important role for the conformation of these oligomers. The 13C chemical shift data of cis and trans proline derivatives, collected in Table 5.29, are useful to determine the stereochemistry of the amino acid-proline bond, e.g. in cyclo-(Pro-Gly)3, melanocyte-stimulating hormone release-inhibiting factor or thyrotropin-releasing hormone. 

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. 

To understand the effects of many trans prolines or of a mixture of cis and trans prolines on the folding kinetics of large proteins, we must consider that cis -> trans isomerizations are intrinsically about 5-10 times faster than trans —cis isomerizations and show small amplitudes only (because the trans state is favored in the unfolded molecules), and that probably not all prolines are important for the folding kinetics. Staphylococcal unclease contains six prolines, but only three of them are rate-limiting for the slow refolding reactions of this protein (Maki et al., 1999). 

Dominidn is a cydic octapeptide isolated from the Caribbean species Eurypon lau lini. This new peptide, whose hill structure has been confirmed by X-ray analysis, contains only amino acids in the l configuration, but has both cis and trans prolines (Williams et al., 2005b). 

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... 

FIGURE 6.24 The cis and trans forms of the amide bond of a dipeptide ester and cyclization of the compound to the piperazine-2,5-dione. The tendency to cyclize is greater when the carboxy-terminal residue is proline or an IV-methylamino acid. In these cases the predominating form is cis, which places the amino and ester groups closer together. 

Most peptide bonds in proteins take on the trans conformation (see p. 66). Only bonds with proline residues (-X-Pro-) can be present in both cis and trans forms. 

Vitagliano, L., Berisio, R., Mastrangelo, A., Mazzarella, L., Zagari, A. Preferred proline puckerings in cis and trans peptide groups implications for collagen stability. Protein Sci. 2001, 10, 2627-32. 

K. Hamacher, Synthesis of n.c.a. cis- and trans-4-[ F]fluoro-L-proline, radiotracers for PET-investigation of disordered matrix protein synthesis, J. Label. Compds Radiopharm. 42 (1999) 1135-1144. 

It has been shown that phosphorylation changes the local conformation of a protein and thereby affects the activity of the complete protein. 341 Phosphorylation of serine and threonine side chains often occurs (Scheme 2). Phosphoamino acids are readily characterized using 3H and 31P NMR experiments. The H and 31P NMR parameters are distinct for phosphorylated serine, threonine, and tyrosine and have also been used to identify both cis-and trans-O-phospho-4-hydroxy-L-proline. 35 Phosphorylation of Cys is rare, but it can be identified by NMR even in large proteins. 36 ... 

Figure 1.3 The peptide bond. Distances are in A (1 A = 0.1 nm). Proline residues are found in both cis and trans conformations.

The conformation of Xaa-Pro peptide bonds in the newly synthesized polypeptide chains prior to cellular folding is not known. The product of protein biosynthesis could be a uniform chain with all peptide bonds in the trans conformation. If this chain starts to fold immediately, then the trom-prolines would be in the correct conformation already, the cis-prolines would be in the incorrect isomeric state, and their trans — cis isomerization would be involved in the folding of all molecules. Alternatively, if there is sufficient time available for the Xaa-Pro bonds of the nascent chains to reach a cis/trans equilibrium (e.g., when folding is transiently arrested by binding to other proteins, such as heat-shock protein (HSP70), then the distribution of prolyl cis and trans isomers prior to cellular folding could be similar to the distribution found in the unfolded protein in vitro. Such a case was encountered in the maturation... 

Asymmetric Michael Addition. An intramolecular Michael reaction catalyzed by (S)-proline leads to the chiral thiadecalin (9) and thiahydrindan (11) and (12). Enone (8) undergoes cyclization in the presence of (S)-proline to give exclusively the trans isomer (9) (eq 4). The thiahydrindandions (11) and (12) are obtained from (10) as a 1 1 mixture of the cis and trans isomers (eq 5). 

Figure 2. Ball and stick representation of the blocked Ala-Pro-Tyr peptide in extended conformation. The aliphatic carbons are labelled according to the lUPAC definition. The cis and trans methylene protons (compared to the a proton) of proline and tyrosine are indexed 1 and 2 respectively. The main backbone and side chain torsional angles referred to in the text are indicated by arrows.

Torsion angles, in addition, may be used to designate the conformation of the side chains. These are denoted by x (x X working along the chain away from Ca). The steric interactions within the side chains in the trans form of the peptide bond (u> = 180°) are much more favorable than those in the cis form (w = 0°), where there may also be steric interference with side chains from residues i- -2. If the residue i+1 is proline, however, the cis and trans forms (Figure 12.25) have similar energies. Proline is the only amino acid taking part in a cis peptide that is normally encountered in proteins. 

When faced with a set of flexible molecules with too large a number of variables, one must turn to chemical modification to reduce the number of variables to a managable set. In the case of peptide hormones, introduction of proline eliminates the torsional degree of freedom < > by introducing a cyclic constraint, but adds the necessity of considering both cis and trans isomers of the amide bond. Replacement of the a-proton of an amino acid by a methyl group has been shown to reduce the number of possible combinations of values for the <(> and i > torsional rotation to essentially two (25). Introduction of a cyclic constraint in a linear flexible molecule reduces the... 

Planar cis and trans isomers (Figure 2.1(a)) are the most stable configurations, because the planar structure involves maximum orbital overlap. For the majority of peptides built up from a-amino acids, the amide bond adopts the trans geometry, a-Imino acids (notably proline but also /V-methylamino acids), as well as a-methyl-a-... 

Recently, Marshall and coworkers have reported on the particular structural behavior of azaproline (azPro) derivative Ac-azPro-NHMe (Fig. 8.3) which tends to form an eight-member hydrogen bond (i — i + 1) in a typical / -turn conformation without introducing any steric bulk. It stabilizes a cis-amide bond whereas proline favors a seven-member H-bond (i + 2 — i) with a trans-amide conformation (y-turn) (Fig. 8.3) [27]. Additional N-methylation decreases the energy difference between cis- and trans-azPro conformers and hence severely perturbs the /fturn conformation [28]. In nonproline derivatives, permethylation inverted the tendency since calculation at the B3LYP/6-31G level showed that the ds-Ac-azAla-NHMe was 1.36 kcal mol-1 more stable than the trans-amide conformer, whereas trans-Ac-azAla-NHMe was favored by 1.24 kcal mol-1 [32]. 

The relevance of catalyzed and spontaneous peptide bond CTI to the biological function of peptides and proteins has inspired considerable effort in research. Thus, distinct pathways have been identified that allow peptide bond isomers to affect physiological signaling differently. To fully understand isomer specificity of bioreactions at the molecular level, it is essential to characterize the structural and electronic differences between cis and trans peptide bond isomers. Most importantly, both isomers cannot sample the same conformational space around proline [30,171], thus presenting an isomer-specific topography to interacting molecules. 

Figure 6. CD spectra of cis- and trans(N,//)-[PtCl(L-prolinate)-(S,S-trans-2-butene)] in acetonitrile (-------------), cis isomer (---), trans isomer.

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. 

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. 

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