Native State Peptide Bond Isomerization

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]. 

Generally, multiple isomerization sites, low propensity of the thermodynamically disfavored isomer, the presence of the isomerization site in flexible protein segments, and poor dispersion of isomer-specific NMR chemical shifts illustrate the difficulties in the detection of native state peptide bond isomerization. However, methods have been developed to characterize native state prolyl isomerization in proteins that deviate much from equal partitioning of isomers [145], 

Spontaneous peptide bond isomerization is relatively slow in respect to the NMR time-scale and, therefore, two resonance frequencies are observable. Nuclear Overhauser effect (NOE) patterns show the NMR couplings between neighboring atoms in space. That is why NOE spectra can be used to discriminate between the resonance patterns for the cis and trans conformers and to calculate the rate of exchange. 

Regulation of biological functions can be achieved via catalytic and binding activities of cellular proteins. In recent years the potential of peptide bond CTI for a switch-like control of protein function beside amino acid side-chain modification or drastically reorientation of a whole polypeptide chains became apparent [147]. 

The best examined proteins that show conformationally heterogeneity in their native states are the sarc homology 2 domain of the interleukin-2 tyrosine kinase (ITK-SH2 domain), the HIV-1 coat protein Gag, the bacteriophage MS2 coat protein, and the transforming growth factor /i-likc domain (TBD) from human fibri-lin-1 [143,148-160]. 

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In the native protein these less stable ds-proline peptides are stabilized by the tertiary structure but in the unfolded state these constraints are relaxed and there is an equilibrium between ds- and trans-isomers at each peptide bond. When the protein is refolded a substantial fraction of the molecules have one or more proline-peptide bonds in the incorrect form and the greater the number of proline residues the greater the fraction of such molecules. Cis-trans isomerization of proline peptides is intrinsically a slow process and in vitro it is frequently the rate-limiting step in folding for those molecules that have been trapped in a folding intermediate with the wrong isomer. 

In native collagen, all Gly-Pro and Xaa-Hyp peptide bonds are in the trans conformation, whereas in the unfolded state, a significant fraction of cis isomers populates at each Gly-Pro and Xaa-Hyp peptide bond, cis-to-trans isomerization reactions at prolyl peptide bonds are the origin for the observed slow kinetics of triple helix formation" as shown by their high activation energy ( 72 kj moG )" and their acceleration by prolyl... 

The fraction of Us molecules depends on the number of proline residues and on their isomeric state in the native protein. In particular, the presence of cts-prolyl peptide bonds in the folded molecules leads to a high fraction of Us, since in unfolded proteins the cis state is populated to a small extent only. Adler and Scheraga (1990) showed by NMR that in heat-unfolded RNase A the nonnative trans isomers predominate at both Pro93 and Proll4. The Up molecules dominate in the unfolded state of proteins that have only tram-prolyl peptide bonds, such as lysozyme (Kato et ai, 1981, 1982), cytochrome c (Ridge el ai, 1981 Nall,... 

In conclusion, native state isomerizations around imidic peptide bonds point to a general role for proline as a molecular switch that can control protein-protein interaction. 

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. 

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. 

Prolyl isomerases are enzymes. In protein folding they catalyze cis trans isomerizations in both directions (Miicke and Schmid, 1992) and show equal efficiencies in unfolding and refolding experiments under identical conditions near the midpoint of the unfolding transition. They carry no information about the isomeric states of the prolyl peptide bonds in the protein substrates. The native isomer is selected by the refolding protein itself simply because the molecules... 

Protein folding can be extremely fast, and some proteins fold to their native state within a few milliseconds. Trans cis peptide bond isomer-izations complicate the folding process and decelerate it, sometimes by more than 1000-fold. Nevertheless, cis peptide bonds occur frequently in folded proteins, mainly before proline and occassionally before other amino acid residues. Prolyl isomerization and conformational folding are coupled Incorrect prolines lower the stability of folding intermediates and partial folding can modulate isomerization rates. Prolyl iso-merases catalyze prolyl isomerizations in protein folding, provided the prolines are accessible. 

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