Proteins folded, prolyl bonds

In native proteins of known three-dimensional structure about 7% of all prolyl peptide bonds are cis (Stewart et al., 1990 MacArthur and Thornton, 1991). Usually, the conformational state of each peptide bond is clearly defined. It is either cis or trans in every molecule, depending on the structural framework imposed by the folded protein chain. There are a few exceptions to this rule. In the native states of staphylococcal nuclease (Evans et al., 1987), insulin (Higgins et al., 1988), and calbindin (Chazin et al., 1989) cis-trans equilibria at particular Xaa-Pro bonds have been detected in solution by NMR. In staphylococcal nuclease, the cis conformer of the Lys 116-Pro 117 bond can be selectively stabilized by bind-... 

Clearly, the action of prolyl isomerases is not restricted to the slow folding of polypeptide chains with intact disulfides, but they also accelerate the oxidative folding of reduced proteins, which resemble more closely the nascent polypeptide chains as they occur in the endoplasmic reticulum. The simultaneous presence of PPI markedly enhances the efficiency of PDI as a catalyst of disulfide bond formation. Both enzymes act according to their specificity and catalyze the isomerization of prolyl peptide bonds and the formation of disulfide bonds, respectively, in the folding protein chains. It remains to be demonstrated that a similar concerted action of the two enzymes can take place in the course of de novo synthesis and folding of proteins in the cell. 

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. 

By use of site-directed mutagenesis in positions covering cis prolyl bonds, the proline has been replaced by nonproline amino acids.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]. 

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. 

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). 

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. 

In crystal structures of folded proteins the prolyl peptide bonds are generally either cis or trans in every molecule. There is, however, an increasing number of exceptions to this rule, and cis/trans equilibria have been found, in particular by 2D-NMR spectroscopy in solution. Examples include staphylococcal nuclease (Evans et al, 1987), insulin (Higgins etal., 1988), calbindin (Chazin et al., 1989 Kordel et al., 1990), scorpion venom Lqh-8/6 (Adjadj et al., 1997), human interleukin-3 (Feng et al., 1997), and the TB6 domain of human fibrillin-1 (Yuan etal., 1997 Yuan et al., 1998). 

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. 

Cis prolines are very well suited to introduce tight turns (such as type VIft turns) into proteins, but this cannot be the sole reason for their widespread occurrence. Evidence increases that cis/trans heterogeneity at prolyl bonds exists not only in the course of protein folding, but also... 

Other important protein-folding catalysts In the ER lumen are peptldyl-prolyl isomerases, a family of enzymes that accelerate the rotation about peptidyl-prolyl bonds in unfolded segments of a polypeptide ... 

Such isomerizations sometimes are the rate-limiting step in the folding of protein domains. Many peptidyl-prolyl isomerases can catalyze the rotation of exposed peptidyl-prolyl bonds indiscriminately in numerous proteins, but some have very specific protein substrates. 

Tertiary amides, such as those associated with prolyl amide bonds frequently influence turn architectures. The importance of the cis Xaa-Pro bond on activity was recognized and proposed to be the source of differentiation in biological activity [86] therefore, isomerization of the prolyl amide bond is central to regulation of protein folding, immunosuppression, and mitosis. These functions are not surprisingly associated with several disease states and thus substitution of the acyl-proline amide bond with the fluoroolefin isostere has received considerable attention. 

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