Refolding kinetics of pig muscle and yeast 3-phosphoglycerate kinases and of their proteolytic fragments.

The time course of refolding of both pig muscle and yeast 3-phosphoglycerate kinase (molecular masses about 47 kDa), as well as their proteolytic C-terminal fragments (30 and 33 kDa, respectively) has been investigated. Very similar refolding kinetics (with half-time between 80-120 s, at 20 degrees C) were observed by fluorescence and ultraviolet absorbance spectroscopy, as well as by activity measurements, for the intact enzyme from both sources. This time course appears not to depend on the time the protein spends in the unfolded state, i.e. it is certainly not controlled by proline isomerization. Furthermore, after removal of a large N-terminal part (molecular mass of about 18 kDa for pig muscle enzyme or 13 kDa for yeast enzyme) of the molecule by proteolysis, refolding of the remaining C-terminal fragment of both proteins follows kinetics virtually indistinguishable from those of the intact protein molecule.

Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe.

Binding of the hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied. It has been shown that ANS has a much stronger affinity to the protein "molten globule" state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, alpha-helical, or beta-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, beta-lactamase, and alpha-lactalbumin as examples.

Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization.

Kinetics of refolding of bovine carbonic anhydrase B have been studied by the "double-jump" technique (i.e. the dependence of protein refolding on delay time in the unfolded state after fast unfolding). It is shown that two stages (the slow with a relaxation time of t1/2 approximately equal to 120 s and the superslow with t1/2 approximately equal to 600 s) observed during refolding of bovine carbonic anhydrase B are due to trans-cis isomerization of proline residues. The dependences of rate constants of these processes on temperature and on the final denaturant concentration were measured. Activation energies of both processes are the same, Ea = 18(+/- 2) kcal/mol. The rate constants of protein refolding do not depend on the final concentration of urea under native conditions. In addition, the rate of isomerization of essential proline residues in the "molten globule" intermediate state of bovine carbonic anhydrase was measured and found to be equal to that for unstructural polypeptides. The effect of several proline residues on carbonic anhydrase refolding is discussed.

Evidence for a molten globule state as a general intermediate in protein folding.

The folding of globular proteins occurs through intermediate states whose characterisation provides information about the mechanism of folding. A major class of intermediate states is the compact 'molten globule', whose characteristics have been studied intensively in those conditions in which it is stable (at acid pH, high temperatures and intermediate concentrations of strong denaturants). In studies involving bovine carbonic anhydrase, human alpha-lact-albumin, bovine beta-lactoglobulin, yeast phosphoglycerate kinase, beta-lactamase from Staphylococcus aureus and recombinant human interleukin 1 beta, we have demonstrated that a transient intermediate which accumulates during refolding is compact and has the properties of the 'molten globule' state. We show that it is formed within 0.1-0.2 s. These proteins belong to different structural types (beta, alpha + beta and alpha/beta), with and without disulphide bridges and they include proteins with quite different times of complete folding (from seconds to decades of minutes). We propose that the formation of the transient molten globule state occurs early on the pathway of folding of all globular proteins.

Correlation between enzyme activity and hinge-bending domain displacement in 3-phosphoglycerate kinase.

Diffuse X-ray-scattering data give evidence for large-scale structural change in pig muscle 3-phosphoglycerate kinase upon substrate binding. Simultaneous binding of 3-phosphoglycerate and MgATP either to the unmodified enzyme or to its active methylated derivative leads to about an 0.1-nm decrease in radius of gyration. These data coincide well with the previous data for yeast 3-phosphoglycerate kinase. When, instead of methylation, the two reactive thiol groups of pig muscle 3-phosphoglycerate kinase are carboxamidomethylated, the enzyme becomes inactive and the radii of gyration of its 'apo' and 'holo' forms do not differ within limits of experimental error. Thus, a correlation exists between the activity of 3-phosphoglycerate kinase and its substrate-induced large-scale conformational change. This correlation is a strong argument in favor of the functional importance of domain locking in the reaction catalyzed by 3-phosphoglycerate kinase.