ABSTRACT
In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils' detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose. The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM). It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases. We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.
