Multy-state protein: Determination of carbonic anhydrase free-energy landscape

Melnik, Bogdan S.; Marchenkov, Victor V.; Evdokimov, S.R.; Samatova, Ekaterina; Kotova, Nina V.

doi:10.1016/j.bbrc.2008.02.096

Cited by 16 publications

(24 citation statements)

References 20 publications

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“…As shown in our previous paper [27], the data on BCA II unfolding obtained by the means of time-resolved fluorescent method under equilibrium conditions agree well with the parameters of chevron plots created by the results of kinetic experiments [30,31]. Thus it has become clear that lifetimes of the excited state of tryptophan residues and its contribution to the steady-state fluorescence intensity at different denaturant concentrations contain information about intermediate states of BCA II.…”

Section: Resultssupporting

confidence: 81%

“…Unfortunately this cannot be achieved using typical spectral methods (for example, steady-state fluorescence and circular dichroism) combined with usual plots of dependence of spectral parameters on the denaturant concentration. Thus, in our previous papers we used dependences of ellipticity and fluorescence intensity at fixed wavelengths on urea concentration to study wild-type BCA II and its mutant forms [29–31]. Those plots allowed us to analyze the presence and stability of intermediate states in each protein (for example, see Fig 1), but they could not explain in what way the mutations affected the folding pathway of this protein.…”

Section: Resultsmentioning

confidence: 99%

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Experimental approach to study the effect of mutations on the protein folding pathway

et al. 2019

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Is it possible to compare the physicochemical properties of a wild-type protein and its mutant form under the same conditions? Provided the mutation has destabilized the protein, it may be more correct to compare the mutant protein under native conditions to the wild-type protein destabilized with a small amount of the denaturant. In general, is it appropriate to compare the properties of proteins destabilized by different treatments: mutations, pH, temperature, and denaturants like urea? These issues have compelled us to search for methods and ways of presentation of experimental results that would allow a comparison of mutant forms of proteins under different conditions and lead to conclusions on the effect of mutations on the protein folding/unfolding pathway. We have studied equilibrium unfolding of wild-type bovine carbonic anhydrase II (BCA II) and its six mutant forms using different urea concentrations. BCA II has been already studied in detail and is a good model object for validating new techniques. In this case, time-resolved fluorescence spectroscopy was chosen as the basic research method. The main features of this experimental method allowed us to compare different stages of unfolding of studied proteins and prove experimentally that a single substitution of the amino acid in three mutant forms of BCA II affected the native state of the protein but did not change its unfolding pathway. On the contrary, the inserted disulfide bridge in three other mutant forms of BCA II affected the protein unfolding pathway. An important result of this research is that we have validated the new approach allowing investigation of the effect of mutations on the folding of globular proteins, because in this way it is possible to compare proteins in the same structural states rather than under identical conditions.

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Experimental approach to study the effect of mutations on the protein folding pathway

et al. 2019

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“…However there was also another circumstance that inspired us to make this research. Having analyzed literature data on the effect of incorporated ss-bridges on the proteins [10]–[16] and having studied the folding of several multi-state proteins [12], [17]–[20], we concluded that the introduction of an ss-bridge affects mainly early intermediate stages upon protein folding, whereas single substitutions of hydrophobic amino acids affect late intermediate stages. Therefore a systematic investigation of the two types of mutations would yield more information on different stages of formation of the structure of multi-state proteins.…”

Section: Resultsmentioning

confidence: 99%

Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

et al. 2012

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The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.

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“…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].…”

Section: Resultsmentioning

confidence: 99%

“…However, the rapidly formed molten globule state affects the parameters characteristic of the following stages of protein folding. The approach that allows taking into account the effect of the fast folding stage of protein folding (occurring during the dead time of the instruments) on the rate of the slow folding stage of this protein is theoretically grounded [1], [14]. This approach permits one to obtain population values of the protein molten globule state versus the denaturant concentration, i.e.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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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Multy-state protein: Determination of carbonic anhydrase free-energy landscape

Cited by 16 publications

References 20 publications

Experimental approach to study the effect of mutations on the protein folding pathway

Experimental approach to study the effect of mutations on the protein folding pathway

Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

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

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