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The cis / trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.

Prolyl cis/trans isomerizations determine the rates of protein folding reactions and can serve as molecular switches and timers. In the gene-3-protein of filamentous phage, Pro-213 trans → cis isomerization in a hinge region controls the assembly of the 2 domains N1 and N2 and, in reverse, the activation of the phage for infection. We elucidated the structural and energetic basis of this proline-limited domain assembly at the level of individual residues by real-time 2D NMR. A local cluster of inter-domain hydrogen bonds, remote from Pro-213, is stabilized up to 3,000-fold by trans → cis isomerization. This network of hydrogen bonds mediates domain assembly and is connected with Pro-213 by rigid backbone segments. Thus, proline cis/trans switching is propagated in a specific and directional fashion to change the protein structure and stability at a distant position.

Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.

The isomerization of the proline peptide bond between tyrosine-92 and proline-93 in bovine pancreatic ribonuclease A has been investigated in the unfolded protein as well as during the slow refolding process. This bond is in the cis state in the native protein. By comparison of various homologous ribonucleases we show that isomerization of proline-93 is associated with a change in fluorescence of tyrosine-92. This provides a spectroscopic probe to monitor this process in the disordered chain after unfolding as well as its reversal in the course of slow refolding. In unfolded ribonuclease incorrect trans isomers of proline-93 are found in both slow-folding species. trans----cis reversal of isomerization of this proline peptide bond during refolding shows kinetics that are identical with the time course of formation of native protein. Isomerization of proline-93 is slower than the formation of a native-like folded intermediate that accumulates on the major slow refolding pathway. Models to explain these results are discussed.