Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Conformational diseases involve the accumulation of misfolded proteins.
• Developing compounds to inhibit toxic protein aggregate formation is crucial.
• Current fluorescent methods for fibril detection lack real-time observation capabilities.

Purpose of the Study:

• To develop a novel method for real-time observation of protein aggregate and fibril formation.
• To utilize green fluorescent protein (GFP) fusions and fluorescence lifetime measurement for studying conformational diseases.
• To investigate the applicability of fluorescence lifetime imaging microscopy (FLIM) for monitoring amyloid-like fibril formation.

Main Methods:

• Recombinant protein expression in E. coli.
• Mass spectrometry for protein primary structure and post-translational modification identification.
• Fluorescent-Lifetime Imaging Microscopy (FLIM) to measure fluorescence lifetime of superfolder green fluorescent protein (SF) and SF-islet amyloid polypeptide (SF-IAPP) fusions in polyacrylamide gel.

Main Results:

• SF-IAPP fusion protein retains the ability to form amyloid-like fibrils.
• SF and SF-IAPP monomers exhibit similar fluorescence lifetime characteristics in gel.
• A significant decrease in average fluorescence lifetime was observed for SF-IAPP within fibrils.

Conclusions:

• FLIM can be applied to measure the average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF).
• This technique offers a promising approach for studying conformational diseases using cellular models.
• The developed method facilitates real-time monitoring of amyloid-like fibril formation.