Rate-limiting step in protein folding

Isomerization of proline residues can he a rate-limiting step in protein folding... 

Enzymes assist formation of proper disulfide bonds during folding Isomerization of proline residues can be a rate-limiting step in protein folding Proteins can fold or unfold inside chaperonins GroEL is a cylindrical structure with a... 

Syn-anti isomerization N-terminal to prolyl peptide fragments is one of the rate limiting steps in protein folding [1], Prolyl fragments exist essentially either in the syn or anti form in native proteins, but as an equilibrium... 

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. 

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. 

Exit from the ER may be the rate-limiting step in the secretory pathway. In this context, it has been found that certain proteins play a role in the assembly or proper folding of other proteins without themselves being components of the latter. Such proteins are called molecular chaperones a number of important properties of these proteins are listed in Table 46—5, and the names of some of particular importance in the ER are listed in Table 46-6. Basically, they stabilize unfolded... 

The reductive nitrosylation of a synthetic iron porphyrin by HNO (193) proceeds with a reported rate constant of 1 x 107 A/-1 s However, this value was estimated based on a HNO dimerization rate constant of 8 x 109 M-1 s-1 (210), which is now considered to be 1000-fold lower [(8 x 106 A/-1 s-1 (106)]. The recalculated constant for the reaction of HNO with the porphyrin (3 x 10s AT V1) is similar to the estimated value of HNO addition to metMb. Synthetic porphyrins generally react 30-fold faster with NO (1 x 109M 1 s-1) than ferrous Mb [for a recent, thorough review see (44)] due to rate-limiting diffusion of NO through the protein. The similarity in rate constants for HNO with metMb and the ferric porphyrin suggests that the rate-limiting step in reductive nitrosylation is likely addition of HNO to the ferric metal, with little influence from the protein structure. 

Most of the small proteins that were used initially as substrates to test the function of prolyl isomerases contained disulfide bonds, which were left intact during unfolding and refolding. These proteins were used because their unfolding is reversible under a wide variety of conditions and because good evidence existed for a number of them that prolyl isomerizations were involved as rate-limiting steps in their slow-folding reactions. A protein chain without disulfides should be a better model... 

With the exception of disulfide bonds, all post-translational modifications must be catalyzed by cellular enzymes. The formation of disulfide bonds can occur at appreciable rates in the absence of enzymes and involves two steps (i) the relatively rapid pairing of cysteines to form S-S bonds that do not correspond to those in the native structure and (ii) disulfide rearrangement (20), The isomerization of disulfide bonds to form the correct cysteine pairs that are present in the native protein is slow and represents an important rate limiting step in folding. For this reason the in-vitro refolding of polypeptides containing several cysteines is usually very slow and inefficient. In eucaryotic cells the formation of the correct disulfide bonds is accelerated by the enzyme protein disulfide isomerase or PDI (38,39), PDI is located... 

At this point it should be noted that not all slow steps in protein folding are prolyl isomerizations. The very slow refolding of large proteins is often limited in rate by other events, such as slow conformational rearrangements, domain-pairing reactions, or subunit associations. An extreme example is provided by the Escherichia coli alkaline phosphatase. This protein requires days to complete folding, but, clearly, this very slow refolding reaction is not related to prolyl isomerization (Dirnbach et al., 1999). 

Figure 6. The sketch of the protein folding pathways. The fast (upper) folding pathway includes the formation of native-like collapsed states In, which rapidly convert into the native state N. The fraction of protein molecules, folding along this pathway, is >. For two-state folders, 1. The lower track (followed by 1 - molecules) represents slow pathway(s), which fold by a three-stage kinetic mechanism. At the first stage, nonspecific collapse species Insc form, which later convert into a collection of discrete native-like intermediates . The transition from Ij to the native state is slow and represents the rate-limiting step in the slow pathway. The degree of heterogeneity in the folding pathways depends on the sequence and external conditions.

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