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].

Strong evidence exists that the translocation of proteins across a variety of membranes involves ti non-native or denatured conformational states. On the other hand a compact state having secondary but not rigid tertiary structure and called the 'molten globule' state has been identified as being stable under mild denaturing conditions. A similar state has been shown to accumulate on the folding pathway of globular proteins. These states are compact though sufficiently expanded to include water, and they are internally mobile. It is proposed that these molten globule states may be suitable candidates for protein translocation across biological membranes.

The background to the concept of the term "molten globule" as a description of intermediates observed in the folding of globular proteins is discussed. These compact intermediates are characterised by certain properties including the presence of secondary structure and considerable conformational mobility compared to the native, functional state. Those intermediates that are thermodynamically stable under mild denaturing conditions have many features in common with the transient intermediates that accumulate significantly during the process of folding. Attention is drawn to cases where the two types are however distinguished on grounds of their Stokes radius, in which cases there is currently no direct evidence for the involvement of the stable intermediates on the folding pathway. Experimental evidence relating to the early stages in folding is reviewed and compared, highlighting the temporal relationship between general collapse of the polypeptide chain and the formation of secondary structure. The continued use of the term "molten globule" is recommended where the minimum essential structural criteria for this state are met.

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