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

Abstract

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.

Emerging Mutations in Nsp1 of SARS-CoV-2 and Their Effect on the Structural Stability

“…It is well known from previous studies that the loop region of the protein plays a critical role in many interactions. Therefore, mutations in these locations may have a particular effect on their structure and thermodynamic properties [ 42 , 43 , 44 , 45 , 46 ].…”

Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy

“…For the fluorescence of bacterial luciferase, lifetime components τ 1 and τ 2 were found to reflect similar information about the stages of protein unfolding ( Table 1 ). It was previously shown that this is not the case for some proteins (see the example of bovine carboanhydrase II [ 21 ]). It should be emphasized that the structural and/or dynamic reasons for the similarities or differences in information based on the fluorescence lifetimes of the protein are not yet understood.…”

“…In addition to information on the stability of the two homologous proteins, we attempted to clarify the question of whether the tryptophan fluorescence lifetime of bacterial luciferase carries new information about the unfolding pathway as compared to widely used steady-state methods. In previous studies, we established that the analysis of fluorescence lifetime allowed for the resolution of two distinct transitions of bovine carboanhydrase II during equilibrium unfolding with urea, which was impossible using other non-kinetic techniques [ 21 ]. One more issue that we endeavored to address is the dependence of conformational stability parameters obtained using fluorescence spectroscopy on the number of the tryptophan residues contained in the protein.…”

A Meat-Derived Lactic Acid Bacteria, Lactobacillus plantarum IIA, Expresses a Functional Parvulin-Like Protein with Unique Structural Property

“…Alternatively, this might imply that Par-LpIIA might be functionally required to accelerate the slow isomerization of cis-proline containing proteins upon folding after newly synthesized from the ribosome. It was reported previously that pH indeed has an effect on the folding rate of proteins (O'Brien et al, 2012;Nemtseva et al, 2019). Rami and Udgaonkar (2001) indicated that the folding rate of protein decreased under extreme alkaline or acidic environment.…”