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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 a-lact-albumin, bovine /3-lactoglobulin, yeast phosphoglycerate kinase, D-lactamase from Staphylococcus aurew and recombinant human interleukin l/I, 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 (8, a+/3 and a/S), 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. Protein folding; Folding intermediate; Folding kinetics; Framework modelThe stable molten globule states obtained under mild denaturing conditions have been shown to be consistently much more compact than state U by viscosity, sedimentation, diffuse X-ray scattering, quasielastic light scattering and urea gradient electrophoresis for CAB [4], ,8Lase [5,6] and aLA [1,2,7]. It is almost as compact as state N [l-7] and has a pronounced secondary structure [l-4,6,8].This secondary structure can be N-like and the molten globule may have some features of the N fold [9]. However, this state differs from state N by the absence of close packing throughout the molecule and by a substantial increase of fluctuations in side chains as well as of larger parts of the molecule [l-3]. In agreement with these data, the equilibrium molten globule states for PLase [lo] and CAB both have Ves on FPLC gel exclusion that are intermediate between the Ves for N and U states. This permits the use of FPLC not only for the evaluation of the compactness of these states but also to monitor the kinetics of the formation of state N in refolding experiments [lo].

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Background: Actinoporins are pore-forming toxins that damage cellular membranes by ␣-helices. Results: An engineered mutant of actinoporin equinatoxin II reveals sequential steps during pore formation. Conclusion: Pore formation is composed of a succession of ordered steps: fast membrane binding followed by the N-terminal region association with the membrane and oligomerization. Significance: Equinatoxin II pore formation does not require stable prepore intermediate as is often found in other poreforming toxins.

Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.All lysosomal and most other proteases are synthesized in the form of inactive precursors (1, 2). Propeptides are generally located N-terminal to the mature enzyme, and activation of the enzyme is accomplished by cis-or trans-cleavage of the propeptide. Propeptides vary from a few (e.g. trypsin) to more than 200 residues (e.g. cathepsin C). Longer propeptides are generally strong and specific inhibitors of their mature enzymes (3-7). In most cases propeptides are also indispensable for correct folding of the enzymes (8). In some enzymes folding of the mature form is extremely slow, and the propeptide assists in overcoming the kinetic barrier (9), which may also be overcome by physicochemical factors such as high ionic strength in subtilisin, for example (10). Proenzymes are quite often more stable than mature enzymes (11, 12) and can represent a pool of latent enzyme until the activation occurs in the proper conditions. Propeptides are also involved in targeting to specific organelles (13, 14); they can affect posttranslational modification such as glycosylation (15) and mediate interactions with other molecules (16,17). Propeptides can be cleaved either by other proteases or by intra-or intermolecular autocatalysis. pH change is one of the most common environmental parameters responsible for triggering the activation of proteases, occurring in cysteine, aspartic acid, and metalloproteases (1,18,19). Low pH is thought either to increase the susceptibility of the propeptide as a substrate due to the protonation of groups close to the cleavage site or to cause a conformational change in the propeptide or enzyme.Cathepsin L is one of the most active cysteine proteases and accounts for most of the lysosomal cysteine protease activity (20). It has been implicated in a range of processes incl...