Molten globule and protein folding.
Explore the source record for details and available documents.
Biological Evolution↗
Biomedical subjects
O B Ptitsyn
Publications and source records attributed to O B Ptitsyn.
How the molten globule became.
Explore the source record for details and available documents.
Animals↗
[The functional state of denatured proteins: the principles of modelling and the first results].
The aqueous environment near cell membranes should be characterized by a local low pH value (due to attraction of protons by the membrane electrostatic potential) and by a local dielectric constant value (due to proximity of the water-organic boundary). It was proposed to simulate the behaviour of proteins near membranes by their behaviour in water-alcohol mixtures at moderately low pH values. Cytochrome c and retinol-binding protein are shown to be denatured at the values of pH and dielectric constant typical for the aqueous environment near the membrane surfaces. The conformational state of both these proteins under mentioned conditions is similar to the molten globule state. The basic function of the retinol-binding protein (transfer and release of vitamin A) is tightly associated with the transition of this carrier protein into the molten globule-like state, as in this case the release of retinol and protein denaturation are coupled and represent a common cooperative process.
Animals↗
"Partly folded" state, a new equilibrium state of protein molecules: four-state guanidinium chloride-induced unfolding of beta-lactamase at low temperature.
Guanidinium chloride- (GdmCl-) induced unfolding of beta-lactamase has been investigated by a combination of size-exclusion chromatography (SEC-FPLC) and usual optical methods. It has been shown that at low temperatures this protein unfolds through two equilibrium intermediates. The first of these intermediates is the molten globule state, while the other (which we have called a "partly folded" state) is less compact than the molten globule but much more compact than the unfolded state. It also preserves a substantial part of secondary structure of the native or molten globule state. We suggest that this new "partly folded" state of a protein molecule can be the equilibrium counterpart of the first kinetic intermediate of protein folding, formed within a few milliseconds, i.e., after the "burst" stage of folding.
Animals↗
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.
Mathematics↗
Kinetic and equilibrium intermediates in protein folding.
Explore the source record for details and available documents.
Kinetics↗
A new approach to artificial and modified proteins: theory-based design, synthesis in a cell-free system and fast testing of structural properties by radiolabels.
A novel approach to the creation of artificial and modified proteins has been elaborated. The approach includes a sequence design based on the molecular theory of protein secondary structure and folding patterns, gene expression in a cell-free system and testing of structural properties of the synthesized polypeptides at a nanogram level using radiolabelled chains. The approach has been applied to a new synthetic protein albebetin which has been designed to form a 3-D fold which does not contradict any structural rule but has been never observed up to now in natural proteins. Using size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis of a radiolabelled chain, it has been shown that the artificial protein is nearly as compact as natural proteins, cooperatively unfolds at high urea concentrations and has some structural features of a definite structure consistent with the designed one. As albebetin has been designed as consisting of two structural repeats, a 'half-albebetin' (one of these repeats) has also been synthesized and studied. It was shown that 'half-albebetin' is also compact.
Amino Acid Sequence↗
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.
Animals↗
Mechanism of pH-induced release of retinol from retinol-binding protein.
A hypothesis is proposed explaining the mechanism of pH-induced release of retinol from retinol-binding protein (RBP). A number of conservative positively charged side chains located on the retinol-binding face of the RBP molecule are involved in salt bridges with conservative negatively charged groups. At low pH these salt bridges are broken and the retinol-binding face of RBP holds from 8 to 12 positively charged groups, which can ensure a proper orientation of the RBP molecule relative to a negatively charged membrane, facilitating the release of retinol. The disruption of salt bridges and the electrostatic repulsion of positive charges can soften the structure of the molecule near the entrance to the retinol-binding pocket, which can trigger both the release of retinol and the transition of RBP to the molten globule state.
Animals↗
'All-or-none' mechanism of the molten globule unfolding.
The Gdm-HCl-induced unfolding of bovine carbonic anhydrase B and S. aureus beta-lactamase was studied at 4 degrees 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.
Carbonic Anhydrases↗
Retinol-binding protein is in the molten globule state at low pH.
Using far- and near-UV circular dichroism, viscosity, tryptophan fluorescence, NMR spectra, binding of a hydrophobic probe, and microcalorimetry, we have shown that the apo form of human retinol-binding protein (RBP) at neutral pH is in a rigid state with properties similar to those of holo-RBP. On the contrary, at acidic pH apo-RBP is in the molten globule state which has been earlier revealed for a number of proteins under mild denaturing conditions. We have also shown that, at equilibrium, the pH-induced retinol release from holo-RBP parallels denaturation of the apoprotein. These findings are consistent with our hypothesis that the transformation of RBP into the molten globule state is involved in the mechanism whereby retinol is delivered to target cells. In particular, a local acidic pH near the membrane surface of target cells might cause the transition of RBP to the molten globule state as well as the release of retinol.
Apoproteins↗
De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.
The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.
Amino Acid Sequence↗
How does protein synthesis give rise to the 3D-structure?
The recent experimental data on stages and kinetic intermediates in protein folding are reviewed. It is emphasized that these data are consistent with the 'framework model' proposed by the author in 1973. The model implies that protein folds by stage mechanism (secondary structure - molten) globule state - native state) in such a way that the results of previous stages are not reconsidered in subsequent ones. Arguments are presented that both these hypotheses and available experimental data do not contradict the assumption that native structures of at least small proteins are nevertheless under thermodynamic rather than kinetic control i.e. correspond to global minima of free energy.
Kinetics↗
Physical reasons for secondary structure stability: alpha-helices in short peptides.
It was recently found that some short peptides (including C- and S-peptide fragments of RNase A) can have considerable helicity in solution, which was considered to be surprising. Does the observed helicity require a new explanation, or is it consistent with previous understanding? In this work we show that this helicity is consistent with the physical theory of secondary structure based on an extension of the conventional Zimm-Bragg model. Without any special modifications, this theory explains reasonably well almost all the experimentally observed dependencies of helicity on pH, temperature, and amino acid replacements. We conclude that the observed "general level" of helicity of C- and S-peptides (5-30% at room temperature and 10-50% near 0 degrees C) is "normal" for short peptides consisting mainly of helix-forming and helix-indifferent residues. The helicity is modified by a multitude of weak specific side chain interactions, many of which are taken into account by the present theory; some discrepancies between the theory and experiment can be explained by weak side-chain-side chain interactions that were neglected. A reasonable coincidence of the theory with experiment suggests that it had been used to investigate the role of local interactions in the formation of alpha-helical "embryos" in unfolded protein chains.
Amino Acid Sequence↗
[Preparation and study of albebetin, an artificial protein with a given spatial structure].
Explore the source record for details and available documents.
Amino Acid Sequence↗
An early immunoreactive folding intermediate of the tryptophan synthease beta 2 subunit is a 'molten globule'.
The refolding kinetics of the tryptophan synthase beta 2 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 beta 2, 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 beta 2, 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 (t1/2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early immunoreactive intermediate recognized by a monoclonal antibody (t1/2 12 s), it is shown that this native-like epitope forms within the 'molten globule', before the tight packing of the protein side chains.
Circular Dichroism↗
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.
Fluorescence↗
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.
Alkylation↗