Prolyl Isomerizations in Protein Folding

Normally, the native protein, N, has each prolyl peptide bond in a unique arrangement, either cis or trans. After unfolding [N— Up, Eq. (1)], however, 

Under solvent conditions that strongly favor folded structure ( strongly native conditions ), chains with certain incorrect isomers can rapidly form intermediates, Ig, which are partially nativelike [Eq. (2)] well before prolyl peptide bond reisomerization occurs (Cook et ai, 1979  

Schmid and Blaschek, 1981 Goto and Hamaguchi, 1982 Kelley et ai, 1986 Kiefhaber et ai, 1990b Nall, 1990). The rate and the extent of this rapid structure formation depends primarily on two factors the location 

The importance of prolyl peptide bond isomerizations for protein folding is indicated by the following experimental observations. 

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, 

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III. Prolyl Isomerizations in Protein Folding A. Fast and Slow Refolding Specks... 

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. 

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... 

The characterization of the molecular nature of rate-limiting steps is a major aim in the elucidation of the folding mechanism of proteins. It is now clear that cis—trans isomerizations of prolyl peptide bonds can be such slow steps. Folding reactions that involve prolyl isomerization were traditionally identified by measuring their kinetic properties and by comparing them with the properties of prolyl isomerization in short peptides (Brandts et ai, 1975 Nall et al., 1978 Schmid and Baldwin, 1978). 

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). 

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... 

The smallest member of a new family of prolyl iso-merases (unrelated to the cyclophilins or the FK-506 binding proteins) that catalyzes the proline-limited folding of a variant of ribonuclease T1 with a KJK value of 30,000 M s With the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide as a substrate in parvulin-catalyzed prolyl isomerization, this parameter is 1.1 x 10 M s Parvulin also accelerates its own refolding in an autocatalytic fashion. 

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