Oleg B. Ptitsyn

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

We propose that the formation of the transient molten globule state occurs early on the pathway of folding of all globular proteins.

The ‘molten globule’ state is involved in the translocation of proteins across membranes?

Strong evidence exists that the translocation of proteins across a variety of membranes involves a 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.

Structures of folding intermediates.

The past year has yielded important results in the study of protein-folding intermediates. It has been shown that the equilibrium molten globule has a native-like tertiary fold and is separated from the unfolded state by a first-order phase transition. New equilibrium intermediates have been revealed and substantial progress has been made in the understanding of two main barriers in protein folding.

Sequential mechanism of refolding of carbonic anhydrase B

The kinetics of refolding of bovine carbonic anhydrase B was studied by a variety of methods over a wide range of times (from milliseconds to hours). It has been shown that protein refolding proceeds through three stages. At the first stage (t ½≈0.03 s) hydrophobic clusters and a compact state of the chain are formed. At the second stage (t ½≈140 s) hydrophobic clusters are desolvated and the rigid native‐like hydrophobic core is formed. At the third stage (t ½≈600 s) the native active protein is formed.

‘Molten‐globule“ state accumulates in carbonic anhydrase folding

Kinetics of folding and unfolding of bovine carbonic anhydrase B were monitored by circular dichroism, viscometry and esterase activity. It was shown that kinetic intermediate states accumulating in folding process reveal a native‐like compactness and secondary structure but have a symmetrized average environment of aromatic side groups and no esterase activity. These properties allow one to consider these intermediate states as the ‘molten‐globule“ state of a protein molecule previously described by us for several equilibrium forms of bovine and human α‐lactalbumins and bovine carbonic anhydrase B.

The molten globule is a third thermodynamical state of protein molecules

Analysis of published data on conformational transitions in relatively small proteins shows that the slopes of these transitions are proportional to the protein molecular weight. It is true both for transitions from the native (N) to the unfolded (U) states (when protein denaturation is coupled to its unfolding) and for transitions from the native to the molten globule (MG) states and from the molten globule to the unfolded state (when protein denaturation is decoupled from protein unfolding). This is precisely the behaviour predicted by thermodynamics for first order phase transitions (‘all‐or‐none’ transitions) in small systems. It follows that N → U, N → MG and MG → U transitions in proteins are all of the ‘all‐or‐none’ type. Thus the molten globule state of protein molecules is separated by an ‘all‐or‐none’ transition both from the native and the unfolded state, i.e. the molten globule state is a third thermodynamic state of protein molecules in addition to the two previously established states ‐ the native and the unfolded.

An early immunoreactive folding intermediate of the tryptophan synthase β2 subunit is a ‘molten globule’

The refolding kinetics of the tryptophan synthase β2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped‐flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured β2, more than half of the protein secondary structure is formed within the dead time of the CD stopped‐flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded β2 the fluorescence of ANS passes through a maximum after about 1 s and then ‘slowly’ decreases. These results show the accumulation, in the 1–10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the ‘molten globule’ state [(1987) FEBS Lett. 224, 9‐13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633‐7640] early imuno‐reactive intermediate recognized by a monoclonal antibody (t 12 s), it is shown that this native‐like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains.

'All-or-none' mechanism of the molten globule unfolding.

The Gdm‐HCl‐induced unfolding of bovine carbonic anhydrase B and S. aureus β‐lactamase was studied at 4°C by a variety of methods. With the use of FPLC it has been shown that within the transition from the molten globule to the unfolded state the distribution function of molecular dimensions is bimodal. This means that equilibrium intermediates between the molten globule and the unfolded states are absent, i.e. the molten globule unfolding follows the ‘all‐or‐none’ mechanism.

Physical nature of the phase transition in globular proteins: Calorimetric study of human α-lactalbumin

The guanidine hydrochloride-induced unfolding of human α-lactalbumin has been studied by isothermal calorimetry. It has been shown that a cooperative transition takes place only in the concentration interval of the denaturant between 0.3 and 2 mol · 1−1. The cooperative transition coincides with the transition detected by circular dichroism in the near-ultraviolet region which reflects the destruction of the specific environment of aromatic side groups. According to scanning calorimetric investigations, the transition disappears in the acid form of the protein where circular dichroism of aromatic side groups is practically absent. At higher concentrations of guanidine hydrochloride, where destruction of the secondary structure and un- folding of the chain are observed, there is no cooperative heat absorption.The guanidine hydrochloride‐induced unfolding of human α‐lactalbumin has been studied by isothermal calorimetry. It has been shown that a cooperative transition takes place only in the concentration interval of the denaturant between 0.3 and 2 mol · 1−1. The cooperative transition coincides with the transition detected by circular dichroism in the near‐ultraviolet region which reflects the destruction of the specific environment of aromatic side groups. According to scanning calorimetric investigations, the transition disappears in the acid form of the protein where circular dichroism of aromatic side groups is practically absent. At higher concentrations of guanidine hydrochloride, where destruction of the secondary structure and un‐ folding of the chain are observed, there is no cooperative heat absorption.

Folding intermediates are involved in genetic diseases

Recent experimental data show that some human genetic diseases are due to mutations in proteins which influence their trafficking and lead to retaining of proteins in the endoplasmic reticulum or their unproper processing. In this paper a hypothesis is proposed that these mutations are connected with an incomplete protein folding, blocking it at the stage of the kinetic molten globule or even earlier. If so, the specific drugs against these diseases may be ligands and other factors which facilitate the correct protein folding.