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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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Refolding experiments.Right panel: Curves of refolding kinetics of apomyoglobin with substitutions A15S and A19S (m2) measured at different urea concentrations with the Trp fluorescence method (emission of 335 nm, excitation of 280 nm). The initial urea concentration was 6.0 M; numbers near the curves indicate final urea concentrations. Solid lines represent single-exponential approximations of the kinetics to zero time. Left panel: Population fI of the molten globule state of protein m2 calculated from the burst-phase fluorescence amplitudes of refolding kinetics. The error of calculated fI does not exceed the size of symbols.
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pone-0098645-g002: Refolding experiments.Right panel: Curves of refolding kinetics of apomyoglobin with substitutions A15S and A19S (m2) measured at different urea concentrations with the Trp fluorescence method (emission of 335 nm, excitation of 280 nm). The initial urea concentration was 6.0 M; numbers near the curves indicate final urea concentrations. Solid lines represent single-exponential approximations of the kinetics to zero time. Left panel: Population fI of the molten globule state of protein m2 calculated from the burst-phase fluorescence amplitudes of refolding kinetics. The error of calculated fI does not exceed the size of symbols.

Mentions: We performed kinetic experiments on mutant forms of apomyoglobin refolding monitored by Trp fluorescence at 335 nm. For example, Fig. 2 (right panel) shows time-resolved courses of the Trp fluorescence changing during refolding of mutant form m2 of apomyoglobin (from 6 M urea to various final urea concentrations). It is seen that at zero time t0 the intensity values of Trp fluorescence at 335 nm are different. This is an indication that within the dead time of the instrument the intermediate state is accumulated. So, at final urea concentrations below 3 M, there are two consecutive refolding phases: The first phase (burst phase) occurs within the dead time of a stopped-flow instrument and is revealed by a jump-wise increase of fluorescence intensity, and the second phase is observed as a slow decrease of fluorescence intensity. At final urea concentrations above 3 M, there is only one fast phase, which manifests itself as a burst-like insignificant increase of fluorescence intensity. So, owing to the instrument dead time, it is only the result of the fast phase (i.e., the transition from the unfolded state to the kinetic intermediate state of apomyoglobin) that can be observed. After the protein, refolding is completed (i.e., time→∞) the fluorescence intensity values correspond to the equilibrium values. It should be noted that the kinetic intermediate I has a higher fluorescence intensity (at 335 nm) than that of the native N or unfolded state U. This property of the intermediate state is used to separate the kinetic transition U↔I from the transition I↔N. Since the slow phase of apomyoglobin refolding always leads to a decrease of fluorescence intensity, folding into the native state is believed to start from the intermediate state. At a given urea concentration M, the transient intermediate state population fI(M) can be calculated from the burst phase amplitude A(M) (see Materials and Methods, and [1]). Baryshnikova et al. [1] described in detail the approach allowing calculating the dependence of the population of the apomyoglobin intermediate state fI(M) on the urea concentration. The gist of the method is that the population of a rapidly formed intermediate state affects the amplitude of the subsequent slow kinetics of folding. For example, Fig. 2 (left panel) demonstrates the population of the molten globule state fI(M) of mutant form m2 of apomyoglobin calculated from the burst phase amplitude kinetic curves in Fig. 2 (right panel) according to Equation 5 (see Materials and Methods, and [1]).


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)

Refolding experiments.Right panel: Curves of refolding kinetics of apomyoglobin with substitutions A15S and A19S (m2) measured at different urea concentrations with the Trp fluorescence method (emission of 335 nm, excitation of 280 nm). The initial urea concentration was 6.0 M; numbers near the curves indicate final urea concentrations. Solid lines represent single-exponential approximations of the kinetics to zero time. Left panel: Population fI of the molten globule state of protein m2 calculated from the burst-phase fluorescence amplitudes of refolding kinetics. The error of calculated fI does not exceed the size of symbols.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4043776&req=5

pone-0098645-g002: Refolding experiments.Right panel: Curves of refolding kinetics of apomyoglobin with substitutions A15S and A19S (m2) measured at different urea concentrations with the Trp fluorescence method (emission of 335 nm, excitation of 280 nm). The initial urea concentration was 6.0 M; numbers near the curves indicate final urea concentrations. Solid lines represent single-exponential approximations of the kinetics to zero time. Left panel: Population fI of the molten globule state of protein m2 calculated from the burst-phase fluorescence amplitudes of refolding kinetics. The error of calculated fI does not exceed the size of symbols.
Mentions: We performed kinetic experiments on mutant forms of apomyoglobin refolding monitored by Trp fluorescence at 335 nm. For example, Fig. 2 (right panel) shows time-resolved courses of the Trp fluorescence changing during refolding of mutant form m2 of apomyoglobin (from 6 M urea to various final urea concentrations). It is seen that at zero time t0 the intensity values of Trp fluorescence at 335 nm are different. This is an indication that within the dead time of the instrument the intermediate state is accumulated. So, at final urea concentrations below 3 M, there are two consecutive refolding phases: The first phase (burst phase) occurs within the dead time of a stopped-flow instrument and is revealed by a jump-wise increase of fluorescence intensity, and the second phase is observed as a slow decrease of fluorescence intensity. At final urea concentrations above 3 M, there is only one fast phase, which manifests itself as a burst-like insignificant increase of fluorescence intensity. So, owing to the instrument dead time, it is only the result of the fast phase (i.e., the transition from the unfolded state to the kinetic intermediate state of apomyoglobin) that can be observed. After the protein, refolding is completed (i.e., time→∞) the fluorescence intensity values correspond to the equilibrium values. It should be noted that the kinetic intermediate I has a higher fluorescence intensity (at 335 nm) than that of the native N or unfolded state U. This property of the intermediate state is used to separate the kinetic transition U↔I from the transition I↔N. Since the slow phase of apomyoglobin refolding always leads to a decrease of fluorescence intensity, folding into the native state is believed to start from the intermediate state. At a given urea concentration M, the transient intermediate state population fI(M) can be calculated from the burst phase amplitude A(M) (see Materials and Methods, and [1]). Baryshnikova et al. [1] described in detail the approach allowing calculating the dependence of the population of the apomyoglobin intermediate state fI(M) on the urea concentration. The gist of the method is that the population of a rapidly formed intermediate state affects the amplitude of the subsequent slow kinetics of folding. For example, Fig. 2 (left panel) demonstrates the population of the molten globule state fI(M) of mutant form m2 of apomyoglobin calculated from the burst phase amplitude kinetic curves in Fig. 2 (right panel) according to Equation 5 (see Materials and Methods, and [1]).

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