China Ocean Aviation Group

Secondary Amide Peptide Bonds

Due to the very small population of cis isomers of secondary amide peptide bonds, often termed nonprolyl peptide bonds, few data exist on the effect of CTI on the chemical and biological properties of polypeptides. In the mid 1970s Ramachan-dran and Mitra calculated a probable appearance for a cis-Ala-Ala peptide bond of 0.1% in the middle position of a tetrapeptide [19]. Experimentally, nonprolyl peptide bonds were found to have a cis content of about 0.5% in dipeptides and of ~0.15% in longer oligopeptides that often serve as references for CTI of unfolded proteins [20,21]. In 1H NMR studies on linear oligopeptides containing alanine residues flanking aromatic amino acids, large isomer-specific differences for the chemical shift of alanyl methyl side-chain have been demonstrated [21]. An upfield shift occurred in the range of 0.3 and 0.5 ppm for the cis isomer of the peptides that allows the calculation of the cis/trans ratio from the integral signal intensities. The free energy difference between the cis and trans isomer is about 11.4 kJ mol-1 for the zwitterionic form of the dipeptide Gly-Gly [20,21]. This mag nitude is similar to the enthalpic difference measured for the N-methyl acetamide conformers (10.7 kJ mol-1, 1.4% cis, D2O) over 30 years ago [10].

Despite this low thermodynamic stability the permanent existence of a single secondary amide peptide bond in cis conformation per 1000 amino acid residues is the minimal population that has to be considered for unfolded polypeptide chains. This cis peptide bond fluctuates across the polypeptide chain in relation to the sequence-specific propensity of a secondary amide peptide bond to adopt the cis conformation. As could be found in folded proteins, nonprolyl cis peptides are frequently located in the b-region of a j/w plot [22]. It was hypothesized that cis peptide bonds represent high-energy structures able to store potential energy for increasing chemical reactivity [23]. Interconversion rates for the reversible CTI of secondary amide peptide bonds typically lead to half times of about 1 s for dipep-tides, which decreases about 4-fold when the peptide bond is positioned in the middle of a longer peptide chain.

Despite the similarity of cis to trans interconversion rates, secondary amide pep-tide bonds and imidic peptide bonds differ greatly in their capacity to form the cis isomeric state. This means that the decreased cis population of secondary amide peptide bonds results from higher rates for CTIs. In the case of the Ala-Tyr dipep-tide a 250-fold difference for the interconversion rate constants was observed (kcfit = 0.6 s-1, ktfic = 2.4 x 10-3 s-1, 298 K) [21]. Generally, chain elongation causes destabilization of the cis conformation when located apart from charged termini, and thus leads to a decreased cis population as well as a lowered barrier to rotation in cis to trans direction.

For example, the Ala-Tyr dipeptide exhibits a AGí value of about 76.7 kJ mol-1 (0.48% cis) whereas the same peptide bond in the pentapeptide Ala-Ala-Tyr-Ala-Ala shows a AGÍ of 64.4 kJ mol-1 (0.11% cis) [21]. The peptide bonds Tyr38-Pro39 of native RNase T1 and Tyr92-Pro93 of native RNase A retained in their cis state when Pro is mutated to Ala. These observations made it possible to compare data for nonprolyl peptide bond isomerization between partially folded protein chains and oligopeptides. Rather similar values were found for the Tyr-Ala CTI in RNase A (kcfit = 0.9-1.4 s-1, 298 K) and RNase T1 (kcfit = 0.702 s-1, ktfic = 2.1 x 10-3 s-1, 288 K) [24,25], and these parameters match rates of Tyr-Ala isomerization in Ala-Ala-Tyr-Ala-Ala (kcfit = 1.77 s-1, ktfic 2.0 x 10-3 s-1 (298 K) [21]. The cis Glu166-Thr167 bond in the cis Pro167Thr TEM-1 b-lactamase variant is also characterized by a rate constant between 1 x 10-3 s-1 and 4 x 10-3 s-1 for the trans to cis interconversion [26]. Therefore the trans to cis isomerization can be rate-limiting in protein folding under native-like conditions, as was shown for a proline-free variant of the a-amylase inhibitor tendamistat [27]. This seems to be proven in the discovery of a novel protein class, the secondary amide peptide cis/trans isomerases (APIases), which can accelerate interconversion of these peptide bonds conformers [28].

Most of the secondary amide cis peptide bonds found in protein structures occur in functionally important regions (e.g. close to the active site of proteins). In the proximity of secondary amide cis peptide bonds the two neighboring Ca (i and i-1) atoms approach one another, enabling p-electron systems of aromatic side-chains to interact with the respective Qb atom. In this case the hydrogen atom of the Ci(5 atom always points to the center of the aromatic ring. This attractive C-H ••• p interaction involves electrostatic dipole-quadrupole polarizabilities. Therefore, in the majority of cases the intimate side-chain/side-chain interactions involve aromatic amino acids [22]. Experimental results with a panel of proline-containing oligopeptides were consistent with a cis isomer stabilizing effect of an aromatic side-chain located N-terminally to the isomerizing bond [29,30], and a similar interaction might be effective for secondary amide peptide bonds too. It was noted that many of the proteins containing nonproline cis peptide bonds were found to be carbohydrate binding or processing proteins [22].

At a nearly unchanged cis/trans ratio, secondary thioxoamide peptide bonds show isomerization rates up to 1000-fold slower when compared with the oxopep-tide congeners. A thioxoamide is an isosteric replacement of the normal peptide bond with only a slight change in the electron distribution of the ground-state. It allows for the photo-switching of cis/trans equilibria of polypeptides under mild photochemical conditions. Irradiation of thioxopeptides in the UV/Vis range leads to a markedly increased cis:trans ratio in the photostationary state [31,32].

0 0

Post a comment