Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II

Nemtseva, Elena V.; Lashchuk, Olesya O; Gerasimova, M. A.; Melnik, Tatiana N.; Nagibina, G. S.; Melnik, Bogdan S.

doi:10.1088/2050-6120/aa994a

Cited by 4 publications

(17 citation statements)

References 30 publications

(87 reference statements)

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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%

“…The double substitution, A53C and A76C, was performed to insert an additional disulfide bridge to BCA II. This mutation stabilized the protein [27]. As demonstrated by the kinetic experiments, each mutation affected the early and later stages of carbonic anhydrase II folding/unfolding in a different way.…”

Section: Resultsmentioning

confidence: 99%

“…As demonstrated by the kinetic experiments, each mutation affected the early and later stages of carbonic anhydrase II folding/unfolding in a different way. The analysis of the chevron plots and rate constants obtained upon nonequilibrium melting of BCA II led to conclusion that the L139A substitution did not change the carbonic anhydrase II folding pathway whereas the disulfide bridge changed it [27].…”

Section: Resultsmentioning

confidence: 99%

“…The second disadvantage that limits fluorescence lifetimes application in this field is the difficulty in connection of obtained discrete lifetimes with the individual tryptophan residues in protein structure. Tryptophan fluorescence decays display, as a rule, three lifetimes independently of whether this amino acid is free in solution or is included into the structure of single- or multi-tryptophan protein [22,23,27,28]. So the fluorescence lifetime components of multi-tryptophan proteins can be assumed as some integral parameters reflecting the structure peculiarities of the protein under the given equilibrium conditions.…”

Section: Resultsmentioning

confidence: 99%

“…In our previous study we demonstrated that application of the fluorescence lifetimes revealed the intermediate states of the model protein during equilibrium denaturation that were “invisible” from the steady-state spectral data [27]. In this work we have used the peculiarities of the time-resolved fluorescent spectroscopy method to study the folding/unfolding pathway of different mutant forms of globular proteins.…”

Section: Introductionmentioning

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%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

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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Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation

Majorina

Melnik

Glukhov

et al. 2022

Front. Mol. Biosci.

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The design of new protein variants is usually confined to slightly “fixing” an already existing protein, adapting it to certain conditions or to a new substrate. This is relatively easy to do if the fragment of the protein to be affected, such as the active site of the protein, is known. But what if you need to “fix” the stability of a protein or the rate of its native or intermediate state formation? Having studied a large number of protein mutant forms, we have established the effect of various amino acid substitutions on the energy landscape of the protein. As a result, we have revealed a number of patterns to help researchers identify amino acid residues that determine the folding rate and the stability of globular proteins states and design a mutant form of a protein with desired properties.

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Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II

Melnik

Katina

Ryabova

et al. 2022

IJMS

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Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).

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Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II

Cited by 4 publications

References 30 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

Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II

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