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Independent of their localization in protein the hydrophobic amino acid residues have no effect on the molten globule state of apomyoglobin and the disulfide bond on the surface of apomyoglobin stabilizes this intermediate state.

Melnik TN, Majorina MA, Larina DS, Kashparov IA, Samatova EN, Glukhov AS, Melnik BS - PLoS ONE (2014)

Bottom Line: In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin.It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin.The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, RAS, Pushchino, Moscow Region, Russia.

ABSTRACT
At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.

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Effect of the SS-bond on apomyoglobin free energy profiles.Free energy profiles estimated for apomyoglobin (WT) and its mutant with a disulphide bond (S-S) between amino acid residues 36 and 106 in 4.3 M urea at pH 6.2, 11°C. N, protein native state; U, protein unfolded state; MG, Intermediate state of a molten globule type; #1, protein N↔MG transition state; #2, protein U↔MG transition state, the height of this energy barrier can not be determined experimentally because the U↔MG transition proceeds during the dead time of the instrument. The error of calculated free energy levels does not exceed 0.5 kJ/mol.
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pone-0098645-g007: Effect of the SS-bond on apomyoglobin free energy profiles.Free energy profiles estimated for apomyoglobin (WT) and its mutant with a disulphide bond (S-S) between amino acid residues 36 and 106 in 4.3 M urea at pH 6.2, 11°C. N, protein native state; U, protein unfolded state; MG, Intermediate state of a molten globule type; #1, protein N↔MG transition state; #2, protein U↔MG transition state, the height of this energy barrier can not be determined experimentally because the U↔MG transition proceeds during the dead time of the instrument. The error of calculated free energy levels does not exceed 0.5 kJ/mol.

Mentions: Fig. 7 shows profiles of free energies for apomyoglobin and its mutant form with the SS-bond estimated from the chevron plot in Fig. 6. One should remember that it is impossible to estimate absolute values of free energies of different states of the protein. We can estimate only the change in the free energy upon transition from one state to the other [21], [31], [57]. In other words, it is possible to estimate how energy levels are located in a protein relative to each other, but it is not always clear how these energy levels of different proteins can be compared. That is why when energy profiles of different proteins or their mutant forms are compared, there is free will in choosing the “reference point” (zero on the energy scale). The choice of such a “reference point” is based usually on additional suppositions. As a rule, it is accepted that free energies of unfolded states of proteins are equal and that unfolded conformations of different proteins are the same and represent statistical coils [1], [24], [31], [58]. The SS-bond introduced into the protein changes the conformation of the protein unfolded state [59]–[61]. Therefore, the disulfide bridge inserted in apomyoglobin has undoubtedly changed the entropy component of the free energy of the unfolded state of this protein [62]; therefore, it would be more correct to level the energy free profiles of apomyoglobin and its mutant form with the SS-bond by the energy of native states of these proteins. This means that to make the comparison more convenient we have attributed the zero value of the free energy in Fig. 7 to the protein native states. Another important moment that ensues from the impossibility to estimate absolute free energies values for different states of the protein is as follows. In the analysis of intricate energy schemes, it is impossible to estimate the stability of a definite state of protein; it is probable to estimate its stability relative to the other state of protein. So it is more correct to speak about the stability of transition between the two states rather than the stability of the state.


Independent of their localization in protein the hydrophobic amino acid residues have no effect on the molten globule state of apomyoglobin and the disulfide bond on the surface of apomyoglobin stabilizes this intermediate state.

Melnik TN, Majorina MA, Larina DS, Kashparov IA, Samatova EN, Glukhov AS, Melnik BS - PLoS ONE (2014)

Effect of the SS-bond on apomyoglobin free energy profiles.Free energy profiles estimated for apomyoglobin (WT) and its mutant with a disulphide bond (S-S) between amino acid residues 36 and 106 in 4.3 M urea at pH 6.2, 11°C. N, protein native state; U, protein unfolded state; MG, Intermediate state of a molten globule type; #1, protein N↔MG transition state; #2, protein U↔MG transition state, the height of this energy barrier can not be determined experimentally because the U↔MG transition proceeds during the dead time of the instrument. The error of calculated free energy levels does not exceed 0.5 kJ/mol.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043776&req=5

pone-0098645-g007: Effect of the SS-bond on apomyoglobin free energy profiles.Free energy profiles estimated for apomyoglobin (WT) and its mutant with a disulphide bond (S-S) between amino acid residues 36 and 106 in 4.3 M urea at pH 6.2, 11°C. N, protein native state; U, protein unfolded state; MG, Intermediate state of a molten globule type; #1, protein N↔MG transition state; #2, protein U↔MG transition state, the height of this energy barrier can not be determined experimentally because the U↔MG transition proceeds during the dead time of the instrument. The error of calculated free energy levels does not exceed 0.5 kJ/mol.
Mentions: Fig. 7 shows profiles of free energies for apomyoglobin and its mutant form with the SS-bond estimated from the chevron plot in Fig. 6. One should remember that it is impossible to estimate absolute values of free energies of different states of the protein. We can estimate only the change in the free energy upon transition from one state to the other [21], [31], [57]. In other words, it is possible to estimate how energy levels are located in a protein relative to each other, but it is not always clear how these energy levels of different proteins can be compared. That is why when energy profiles of different proteins or their mutant forms are compared, there is free will in choosing the “reference point” (zero on the energy scale). The choice of such a “reference point” is based usually on additional suppositions. As a rule, it is accepted that free energies of unfolded states of proteins are equal and that unfolded conformations of different proteins are the same and represent statistical coils [1], [24], [31], [58]. The SS-bond introduced into the protein changes the conformation of the protein unfolded state [59]–[61]. Therefore, the disulfide bridge inserted in apomyoglobin has undoubtedly changed the entropy component of the free energy of the unfolded state of this protein [62]; therefore, it would be more correct to level the energy free profiles of apomyoglobin and its mutant form with the SS-bond by the energy of native states of these proteins. This means that to make the comparison more convenient we have attributed the zero value of the free energy in Fig. 7 to the protein native states. Another important moment that ensues from the impossibility to estimate absolute free energies values for different states of the protein is as follows. In the analysis of intricate energy schemes, it is impossible to estimate the stability of a definite state of protein; it is probable to estimate its stability relative to the other state of protein. So it is more correct to speak about the stability of transition between the two states rather than the stability of the state.

Bottom Line: In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin.It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin.The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, RAS, Pushchino, Moscow Region, Russia.

ABSTRACT
At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.

Show MeSH