Gennady V. Semisotnov

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.

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.

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.

Secondary structure of globular proteins at the early and the final stages in protein folding

The ellipticities for an early transient intermediate in refolding observed by kinetic circular dichroism measurements at 220–225 nm for 14 different proteins are summarized, and the ellipticity values are compared with those for the final native proteins and also with the ellipticities expected from a physical theory of protein and polypeptide secondary structure. The results show that a substantial part of the protein secondary structure is in general formed in the earliest detectable intermediate in refolding and that the ellipticities in both the native and the intermediate states are consistent with the physical theory of protein secondary structure.

Three-state protein folding: Experimental determination of free-energy profile

When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three‐state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea‐dependent rates of the I↔N and U↔N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.

Molecular chaperone GroEL/ES: Unfolding and refolding processes

Molecular chaperones are a special class of heat shock proteins (Hsp) that assist the folding and formation of the quaternary structure of other proteins both in vivo and in vitro. However, some chaperones are complex oligomeric proteins, and one of the intriguing questions is how the chaperones fold. The representatives of the Escherichia coli chaperone system GroEL (Hsp60) and GroES (Hsp10) have been studied most intensively. GroEL consists of 14 identical subunits combined into two interacting ring-like structures of seven subunits each, while the co-chaperone GroES interacting with GroEL consists of seven identical subunits combined into a dome-like oligomeric structure. In spite of their complex quaternary structure, GroEL and GroES fold well both in vivo and in vitro. However, the specific oligomerization of GroEL subunits is dependent on ligands and external conditions. This review analyzes the literature and our own data on the study of unfolding (denaturation) and refolding (renaturation) processes of these molecular chaperones and the effect of ligands and solvent composition. Such analysis seems to be useful for understanding the folding mechanism not only of the GroEL/GroES complex, but also of other oligomeric protein complexes.

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

GroES co‐chaperonin small‐angle X‐ray scattering study shows ring orifice increase in solution

GroES consists of seven identical 10 kDa subunits and is involved in assisting protein folding as the partner of another oligomeric protein, the GroEL chaperonin. Here we studied the GroES structure in solution using small‐angle X‐ray scattering (SAXS). The SAXS pattern, calculated for the GroES crystal structure, was found to be different from the experimental one measured in solution. The synchronic shift in the radial direction and some turning of the protein subunits eliminate the difference and result in the increase of the hole diameter in the GroES ring‐like structure from 8 Å in the crystal to 21 Å in solution.

GroEL-Assisted Protein Folding: Does It Occur Within the Chaperonin Inner Cavity?

The folding of protein molecules in the GroEL inner cavity under the co-chaperonin GroES lid is widely accepted as a crucial event of GroEL-assisted protein folding. This review is focused on the data showing that GroEL-assisted protein folding may proceed out of the complex with the chaperonin. The models of GroEL-assisted protein folding assuming ligand-controlled dissociation of nonnative proteins from the GroEL surface and their folding in the bulk solution are also discussed.