Publisher Summary This chapter describes the present state of the studies of the molten globule state and its role in protein folding and physiological processes Recent data on the intermediates (equilibrium and kinetic), focusing attention on the molten globule state are discussed in the chapter The molten globule has a native-like overall architecture (folding pattern) without including the rigid packing of side chains It is separated by first-order phase transitions from both the native and the unfolded states and represents a third thermodynamic state of protein molecules It is a universal kinetic intermediate in protein folding; the structure of this intermediate is similar to the structure of the equilibrium molten globule It is predicted and confirmed experimentally that the molten globule state can exist in a living cell and plays an important role in a number of physiological processes

Molten globule and protein folding.

The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.

Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding

Hydrogen exchange pulse labeling and stopped-flow circular dichroism were used to establish that the structure of the earliest detectable intermediate formed during refolding of apomyoglobin corresponds closely to that of a previously characterized equilibrium molten globule. This compact, cooperatively folded intermediate was formed in less than 5 milliseconds and contained stable, hydrogen-bonded secondary structure localized in the A, G, and H helices and part of the B helix. The remainder of the B helix folded on a much slower time scale, followed by the C and E helices and the CD loop. The data indicate that a molten globule intermediate was formed on the kinetic folding pathway.

https://science.sciencemag.org/content/sci/262/5135/892.full.pdf

Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin

Proteins, due to the delicate balance of stabilizing and destabilizing interactions, are only marginally stable. Adaptation to extreme environments tends to shift the ‘mesophilic’ characteristics of proteins to the respective extremes of temperature, hydrostatic pressure, pH and salinity, such that, under the mutual physiological conditions, the molecular properties are similar regarding overall topology, flexibility and solvation. Enhanced intrinsic stability requires only minute local structural changes so that general strategies of stabilization cannot be established. Apart from mutative changes of amino-acid sequences, extrinsic factors (or cellular components) may be involved in ‘extremophilic adaptation’.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1991.tb16426.x

Protein stability and molecular adaptation to extreme conditions.

Publisher Summary This chapter provides an insight that quantitative predictions of the stability and cooperative folding behavior of proteins are within reach. The problem of predicting the structure of a protein from its amino acid sequence constitutes the classical “protein folding problem.” The thermodynamic aspects of this problem constitute the focus of the chapter. Experimental thermodynamic values for the fundamental forces that determine protein stability are refined to a point in which accurate calculations of the free energy of stabilization are possible, provided that the protein structure is known. The chapter also (1) predicts the overall stability of proteins based on structural information, (2) predicts the cooperative folding/unfolding behavior of proteins, (3) predicts the magnitude of the interactions among structural domains in proteins, and (4) predicts the presence of specific partially folded intermediates. Estimation of the folding/unfolding partition function using a hierarchical approximation and subsequent prediction of cooperative behavior is also increasingly feasible, as demonstrated for myoglobin and yeast phosphoglycerate kinase. Thermodynamic stability and cooperative interactions in proteins can be determined by hierarchical approach to partition functions, thermodynamic dissection of cooperative interactions, and classification of cooperative forces and mechanisms. The strategy used to dissect specific fundamental interactions is to isolate first the hydrophobic effects and subsequently other interactions. Some future work will extend the capability to proteins exhibiting other than pure α-helical structural motifs.

Thermodynamics of structural stability and cooperative folding behavior in proteins.

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.

R. H. Pain

Papers

Molten globule intermediates and protein folding.