DYNAMICS OF CONFORMATIONAL CHANGES IN FLUORESCENTLY LABELED G-ACTIN UPON INTERACTION WITH CYTOCHROME C AS EVIDENCE OF POTENTIAL INVOLVEMENT OF CYTOSKELETAL PROTEINS IN APOPTOSIS

Existing data suggest the involvement of cytoskeletal proteins, particularly actin, in the process of apoptosis. However, the exact role of actin remains unclear. The aim of this study was to investigate the potential functional interaction of this protein with one of the key participants in apoptosis, cytochrome c. We utilized fluorescent labeling of amino acid residues of rat brain-derived b/g-actin in its monomeric form (G-actin) using the fluorescent dye 3-(4-carboxybenzoyl) quinoline2-carboxaldehyde (CBQCA) to monitor induced molecular changes. It is well known that CBQCA reacts with primary amines in the presence of cyanide to form a fluorescent product. However, we also discovered the formation of another fluorescent derivative when the dye reacted with the phenolic group of free tyrosine, as well as its residues in peptides and proteins, independent of cyanide presence. During the investigation of conformational changes in labeled G-actin molecules, we found that the fluorescence intensity varied in the spectral region characteristic of the reaction product with tyrosine residues, but not with amine groups. Specifically, cytochrome c at micromolar concentrations decreased fluorescence in a dose-dependent manner, indicating direct interaction with G-actin. Actin-specific toxins and other factors affecting its polymerization also reduced the fluorescence intensity of labeled actin, without significantly influencing the effect of cytochrome c. At the same time, cytochrome c had no effect on the fluorescence of inactivated labeled actin. Thus, by utilizing the previously undescribed CBQCA labeling of tyrosine residues, we were able to study conformational changes in the actin molecule and establish the interaction of this protein with cytochrome c. It can be hypothesized that actin is involved in apoptosis as a regulator of apoptosome assembly, particularly through its interaction with cytochrome c.