Native State Isomerization

Simultaneous Action of Prolyl Isomerase and Protein Disulfide-Isomerase as Catalysts of Folding 

At least two major slow processes occur in the folding of disulfide-containing proteins the cis-trans isomerizations of Xaa-Pro peptide bonds and the formation of the correct disulfide bonds. The latter is catalyzed by protein disulfide-isomerase (PDl). This enzyme occurs at high concentration in the endoplasmic reticulum (Hawkins et al., 1991) and there is good experimental evidence that PDl is required for the de novo folding of nascent secretory proteins (Bulleid and Freedman, 1988). Cyclophilins have recently also been localized in the ER (Hasel et al., 1991) and in other compartments of the secretory pathway (Caroni et al., 1991). Their biological function is not known. 

In in vitro experiments prolyl isomerase accelerates the oxidative folding of reduced RNase T1 (i.e., folding coupled with formation of the disulfide bonds) and the catalysis of disulfide bond formation by PDl is markedly improved when PPI is present simultaneously (Schonbrunner and Schmid, 1992). The oxidative folding of RNase T1 in the presence of a mixture of reduced and oxidized glutathione is a slow process and it can be followed by the increase in tryptophan fluorescence (Fig. 7). Folding is strictly linked to disulfide bond formation under the conditions 

An analogous effect of prolyl isomerase is noted when disulfide bond formation is catalyzed by disulfide isomerase. In the absence of prolyl 

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. 

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In the given examples of native state isomerization for ITK and MS2 the trans form corresponds to an extended, solvent-exposed loop conformation. Cis forms are characterized by extensive contacts between the loops and the surface of the proteins compared to the trans conformers, where those contacts are weak or not present. It is speculated that such contacts provide the necessary energy needed to stabilize the inherently less stable cis conformation [147]. 

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. 

Other evidence for different classes of proline residues has come from energy calculations (Levitt, 1981 Ihara and Ooi, 1985), in which the destabilization of the native state was calculated when one proline at a time was incorporated, in its incorrect isomeric state, into the protein. Levitt (1981) classified these proline residues into three categories. Type I prolines destabilize the native state only to a small extent when in the incorrect isomeric state. Such prolines should barely affect folding. 

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],... 

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. 

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. 

First evidence for a regulatory function of native-state prolyl isomerization comes from the parvulins Pinl and Essl, which are specific for pSer/pThr-Pro sequences and possibly link regulation by prolyl isomerization to regulation by phosphorylation/dephosphorylation. Similarly, the correlation between prolyl isomerization and Ca2+ binding in the mammalian lectins might indicate that prolyl isomerization could also be connected with Ca2+ signaling. 

In the future we need to elucidate which of the many interactions between prolyl isomerases and target proteins are functionally important in the cell and how prolyl isomerase domains of large proteins contribute to the overall functions of these proteins. At the molecular level the kinetic and structural consequences of native-state prolyl isomerization must be elucidated at high resolution to understand how these isomerizations can contribute to cellular regulation, and how they are modulated by prolyl isomerases and by other processes, such as phosphorylation. 

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 Photoactive Yellow Protein (PYP) is the blue-light photoreceptor that presumably mediates negative phototaxis of the purple bacterium Halorhodospira halophila [1]. Its chromophore is the deprotonated trans-p-coumaric acid covalently linked, via a thioester bond, to the unique cystein residue of the protein. Like for rhodopsins, the trans to cis isomerization of the chromophore was shown to be the first overall step of the PYP photocycle, but the reaction path that leads to the formation of the cis isomer is not clear yet (for review see [2]). From time-resolved spectroscopy measurements on native PYP in solution, it came out that the excited-state deactivation involves a series of fast events on the subpicosecond and picosecond timescales correlated to the chromophore reconfiguration [3-7]. On the other hand, chromophore H-bonding to the nearest amino acids was shown to play a key role in the trans excited state decay kinetics [3,8]. In an attempt to evaluate further the role of the mesoscopic environment in the photophysics of PYP, we made a comparative study of the native and denatured PYP. The excited-state relaxation path and kinetics were monitored by subpicosecond time-resolved absorption and gain spectroscopy. 

The early stages of folding of barstar have been measured on the microsecond time scale by temperature jumping its cold-denatured state from 2 to 10°C.65,66 There is the fast formation of a folding intermediate (tm 200 fxs) with the peptidy 1-proline 48 bond trans, followed by the formation with ty2 60 ms of a second intermediate that is highly native-like because it binds to and inhibits barnase. The native-like intermediate then undergoes trans cis peptidyl-proline isomerization on the time scale of minutes to give the final native structure (equation 19.2). 

The beanlike seeds of the trees and shrubs of the genus Erythrina, a member of the legume family, contain substances that possess curare-like activity. The plants are widely distributed in the tropical and subtropical areas of the American continent, Asia, Africa, and Australia, but apparently they are not used by the natives in the preparation of arrow poisons. Of 105 known species, the seeds from more than 50 have been tested, and all were found to contain alkaloids with curariform properties. Erythroidine, from E. americana, was the first crystalline alkaloid of the group to be isolated. It consists of at least two isomeric alkaloids, a and P-erythroidine both are dextrorotatory. Most experimental and clinical study has centered on the b form because it is more readily obtainable in pure state. P-Erythroidine is a tertiary nitrogenous base. Several hydrogenated derivatives of p-erythroidine have been prepared of these, dihydro-P-erythroidine has been studied most carefully and subjected to clinical trial. Conversion of P-erythroidine into the quaternary metho salt (p-erythroidine methiodide) does not enhance, but rather almost entirely, abolishes its curariform activity this constitutes a notable exception to the rule that conversion of many alkaloids into quaternary metho salts results in the appearance of curare-like action. 

A final concern is the extent to which the proportion of each conformer is faithfully captured by the native gel. Although very small structural differences, such as isomerization within an active site, may not be resolved unless they alter the hydrodynamic profile of the RNA, native PAGE results generally correlate well with other measures of RNA folding in solution. Because 10-30 s are needed for samples to enter the gel, native PAGE is most successful at resolving conformational states that do not exchange within this time (Fig. 9.3A). The entrapment of different conformers is... 

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,... 

Scheme II. Kinetic model for the slow-refolding reactions of RNase T1 under strongly native conditions, U stands for unfolded species, I for intermediates of refolding, and N is the native protein. The superscript and the subscript indicate the isomeric states of Pro39 and Pro55, respectively, in the correct, nativelike cis (c) and in the incorrect, nonnative trans (t) isomeric state. As an example, 155 stands for an intermediate with Pro55 in the correct cis and Pro39 in the incorrect trans state. The time constants given for the individual steps refer to folding conditions of 0.15 M GdmCl, 0.1 M Tris-HCl, pH 8.0, at 10°C. From Kiefhaber et al. (1990b,c).

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