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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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Urea induced equilibrium unfolding of apomyoglobin mutants.Equilibrium unfolding of apomyoglobin (WT) and its mutant forms m2 – A15S, A19S; m3 - A15S, A19S, V21T; m4 – L9D, A15S, A19S, V21T; m6 – L9D, A15S, A19S, V21T, V66T, A74S) measured with the method of Trp fluorescence. Native state population was calculated as a normalized value of the relation of fluorescence intensities at wavelengths of 320 nm and 380 nm (I320/I380).
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pone-0098645-g004: Urea induced equilibrium unfolding of apomyoglobin mutants.Equilibrium unfolding of apomyoglobin (WT) and its mutant forms m2 – A15S, A19S; m3 - A15S, A19S, V21T; m4 – L9D, A15S, A19S, V21T; m6 – L9D, A15S, A19S, V21T, V66T, A74S) measured with the method of Trp fluorescence. Native state population was calculated as a normalized value of the relation of fluorescence intensities at wavelengths of 320 nm and 380 nm (I320/I380).

Mentions: The kinetics of refolding for all mutant forms of apomyoglobin were measured and the populations of the molten globule state were calculated. Fig. 3 shows dependencies of populations fI of the molten globule state versus urea concentration for all mutant forms of apomyoglobin with substitutions of hydrophobic amino acid residues on the protein surface and in its hydrophobic core. Table 1 lists values of urea concentration corresponding to the midpoint of transition MG↔U (Cm,MG↔U) for wild type apomyoglobin (WT) and its mutant forms. As can be seen, none of these substitutions have effect on the stability of the molten globule state. But all mutations change the stability of the native state of apomyoglobin. This can be concluded from the curves of equilibrium unfolding of the mutant forms of apomyoglobin. Fig. 4 demonstrates the curves of equilibrium unfolding for the four mutant proteins with substitutions of hydrophobic amino acid residues on the surface of apomyoglobin. The equilibrium unfolding study of the mutant forms of apomyoglobin with amino acid residue substitutions in the protein hydrophobic core was described in detail in our previous study [15], [48]–[49]. It should be noted that urea-induced equilibrium unfolding curves of apomyoglobin represent three-state transition [1]. This means that transition from native state N to unfolded state U contains two transitions N↔MG and MG↔U, which cannot be separated. Table 1 lists the data on equilibrium unfolding of the mutant forms of apomyoglobin with hydrophobic residue substitutions both on the protein surface and in its hydrophobic core. Table 1 shows that the urea concentration values corresponding to the midpoint of equilibrium unfolding Cm,N↔U differ for all mutant forms of apomyoglobin, and it is seen that all studied mutations destabilized transition N↔U. The urea concentration values corresponding to the midpoint of population of the molten globule state Cm,MG↔U are the same within the error. Thus, all the studied hydrophobic amino acid residue substitutions affected the native state of apomyoglobin to a different extent, but did not change the stability of the molten globule state. As Cm,N↔U is dependent on two transitions (N↔MG and MG↔U), the fact that for some mutants, e.g. L61A, m4 and m6, the Cm,N↔U values calculated from equilibrium unfolding (fig. 4), are lower than the Cm,MG↔U values calculated from population curves (Fig. 3) means that the transition from native state N to molten globule state MG proceeds at urea concentrations much lower than those upon transition from molten globule state to unfolded state U. This circumstance is not surprising and is observed for many proteins the unfolding of which results in accumulation of intermediate states. In other words, when the denaturant concentration increases, the protein first passes from native state N to intermediate state I, and then from intermediate state I to unfolded state U. It is evident that transition N↔I is less stable than transition I↔U. This takes place, for example, in some mutant forms of apomyoglobin studied in detail [11] as well as in carboxyanhydrase [14], lipase [50] and other proteins [51], [52].


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)

Urea induced equilibrium unfolding of apomyoglobin mutants.Equilibrium unfolding of apomyoglobin (WT) and its mutant forms m2 – A15S, A19S; m3 - A15S, A19S, V21T; m4 – L9D, A15S, A19S, V21T; m6 – L9D, A15S, A19S, V21T, V66T, A74S) measured with the method of Trp fluorescence. Native state population was calculated as a normalized value of the relation of fluorescence intensities at wavelengths of 320 nm and 380 nm (I320/I380).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098645-g004: Urea induced equilibrium unfolding of apomyoglobin mutants.Equilibrium unfolding of apomyoglobin (WT) and its mutant forms m2 – A15S, A19S; m3 - A15S, A19S, V21T; m4 – L9D, A15S, A19S, V21T; m6 – L9D, A15S, A19S, V21T, V66T, A74S) measured with the method of Trp fluorescence. Native state population was calculated as a normalized value of the relation of fluorescence intensities at wavelengths of 320 nm and 380 nm (I320/I380).
Mentions: The kinetics of refolding for all mutant forms of apomyoglobin were measured and the populations of the molten globule state were calculated. Fig. 3 shows dependencies of populations fI of the molten globule state versus urea concentration for all mutant forms of apomyoglobin with substitutions of hydrophobic amino acid residues on the protein surface and in its hydrophobic core. Table 1 lists values of urea concentration corresponding to the midpoint of transition MG↔U (Cm,MG↔U) for wild type apomyoglobin (WT) and its mutant forms. As can be seen, none of these substitutions have effect on the stability of the molten globule state. But all mutations change the stability of the native state of apomyoglobin. This can be concluded from the curves of equilibrium unfolding of the mutant forms of apomyoglobin. Fig. 4 demonstrates the curves of equilibrium unfolding for the four mutant proteins with substitutions of hydrophobic amino acid residues on the surface of apomyoglobin. The equilibrium unfolding study of the mutant forms of apomyoglobin with amino acid residue substitutions in the protein hydrophobic core was described in detail in our previous study [15], [48]–[49]. It should be noted that urea-induced equilibrium unfolding curves of apomyoglobin represent three-state transition [1]. This means that transition from native state N to unfolded state U contains two transitions N↔MG and MG↔U, which cannot be separated. Table 1 lists the data on equilibrium unfolding of the mutant forms of apomyoglobin with hydrophobic residue substitutions both on the protein surface and in its hydrophobic core. Table 1 shows that the urea concentration values corresponding to the midpoint of equilibrium unfolding Cm,N↔U differ for all mutant forms of apomyoglobin, and it is seen that all studied mutations destabilized transition N↔U. The urea concentration values corresponding to the midpoint of population of the molten globule state Cm,MG↔U are the same within the error. Thus, all the studied hydrophobic amino acid residue substitutions affected the native state of apomyoglobin to a different extent, but did not change the stability of the molten globule state. As Cm,N↔U is dependent on two transitions (N↔MG and MG↔U), the fact that for some mutants, e.g. L61A, m4 and m6, the Cm,N↔U values calculated from equilibrium unfolding (fig. 4), are lower than the Cm,MG↔U values calculated from population curves (Fig. 3) means that the transition from native state N to molten globule state MG proceeds at urea concentrations much lower than those upon transition from molten globule state to unfolded state U. This circumstance is not surprising and is observed for many proteins the unfolding of which results in accumulation of intermediate states. In other words, when the denaturant concentration increases, the protein first passes from native state N to intermediate state I, and then from intermediate state I to unfolded state U. It is evident that transition N↔I is less stable than transition I↔U. This takes place, for example, in some mutant forms of apomyoglobin studied in detail [11] as well as in carboxyanhydrase [14], lipase [50] and other proteins [51], [52].

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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