The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure.

Publisher Summary This chapter deals with the structure of the molten globules of various globular proteins revealed by the recent experimental studies. Recent advances in experimental techniques, including hydrogen-exchange NMR, solution X-ray scattering, and protein engineering, have provided detailed pictures of the molten globules for these proteins. The molten globule state has heterogeneous structures, in which one portion of a molecule is more organized and native-like with the other portions being less organized, although the overall structure satisfies the criteria of the molten globule state (compactness, the presence of secondary structure, and the lack of rigid tertiary structure). The chapter describes how the molten globule state has been identified as the intermediate of kinetic refolding and discusses the kinetic roles of the molten globule state in protein folding. The chapter also discusses thermodynamic stability and cooperativity of the molten globule state from the viewpoint of the hierarchy of protein folding, in which the molten globule state plays a role as a junction of two levels of the hierarchy.

Role of the molten globule state in protein folding.

Thermodynamic study of the apomyoglobin structure

The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak to peak distances. The r values have been found to be not only related to the tyrosine/tryptophan ratio but also dependent on the polarity of the medium in which tyrosyl residues are embedded. The results obtained on purified proteins have been found consistent with the available X-ray information and with the existing solvent perturbation data.

Determination of tyrosine exposure in proteins by second-derivative spectroscopy.

Second derivative spectroscopy has been used to resolve the complex protein spectrum in the near-ultraviolet region and the contributions of the three aromatic chromophores have been evaluated. A method for the direct quantitative determination of phenylalanine and tryptophan in proteins has been carried out. Phenylalanine determination has been carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination has been performed in the 290-295-nm region and the experimental values have been corrected for the presence of tyrosine. The results obtained on 10 highly purified proteins have been found in good agreement with those obtained from sequence analysis.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1978.tb12622.x

Second-Derivative Spectroscopy of Proteins A Method for the Quantitative Determination of Aromatic Amino Acids in Proteins

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2876%2980585-6

Equilibrium evidence of non-single step transition during guanidine unfolding of apomyoglobins.

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.

Myoglobin structure and regulation of solvent accessibility of heme pocket

1. The heart ventricle myoglobin of Atlantic bluefin tuna has been purified and its amino acid composition has been determined. 2. The perturbing effect of guanidine hydrochloride on the molecular structure of tuna ferrimyoglobin and its corresponding apoprotein has been investigated by Soret absorbance and ultraviolet fluorescence. 3. The conformation-free energy of unfolding delta G0 has been calculated by thermodynamic treatments of the data concerning guanidine unfolding. 4. The results have been compared with other known myoglobins, particularly those of yellowfin tuna.

Amino acid composition and physico-chemical properties of bluefin tuna (Thunnus thynnus) myoglobin.

1. 1. The skeletal muscle myoglobin of the water buffalo ( Bos bubalus L.) has been purified by a rapid and simplified procedure. 2. 2. The amino acid composition of the myoglobin has been determined. 3. 3. The N -terminal amino acid is glycine. 4. 4. The results have been compared with other mammalian myoglobins, particularly that of the cow.

The skeletal muscle myoglobin of the water buffalo (Bos bubalus L.)

The free energy of unfolding of several myoglobins from different animal species has been determined from their denatunation pattern by using the ligand binding model. The results indicate that no simple correlation exists between the free energy of unfolding of myoglobin and the basal metabolic rate of the animal species from which the myoglobin was isolated

Gaetano Irace

Papers

Equilibrium evidence of non-single step transition during guanidine unfolding of apomyoglobins.

Myoglobin structure and regulation of solvent accessibility of heme pocket

Amino acid composition and physico-chemical properties of bluefin tuna (Thunnus thynnus) myoglobin.

The skeletal muscle myoglobin of the water buffalo (Bos bubalus L.)

Conformational stability and basal metabolic rate: reexamination of the case of myoglobin