Teikichi Ikura

Effects of Proline Mutations on the Folding of Staphylococcal Nuclease

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.

Denaturation and reassembly of chaperonin GroEL studied by solution X-ray scattering.

We measured the denaturation and reassembly of Escherichia coli chaperonin GroEL using small‐angle solution X‐ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea‐induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil‐like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X‐ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same structure and the molecular mass as the native GroEL. These results demonstrate that the fully unfolded GroEL monomer can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis and preexisting chaperones. Therefore, GroEL can, in principle, fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under in vivo conditions, in order to avoid misfolding and aggregation.

Solution X-ray scattering study on the chaperonin GroEL from Escherichia coli

The molecular architecture of native GroEL has been studied by solution X-ray scattering. The radius of gyration for the native molecule was estimated to be 66.0 A in 50 mM Tris-HCl, 100 mM KCl at pH 7.5 and 25 degrees C. The maximum dimension was estimated to be 170 A, based on the pair distance distribution function. A cylindrical structure or two heptameric rings was found to be the best for native GroEL among structures examined by using a multi-sphere model analysis in which the radius of constituent sphere was 6 A. The results of the model analysis show that the radius of GroEL is 68.0 A and the height is 150.7 A. Unexpectedly, the central penetrating hole through GroEL was not confirmed in the best-fit structure.