Related Experiment Video
Updated: Jul 7, 2026
Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging
Published on: March 27, 2020
Effect of an amyloidogenic sequence attached to yellow fluorescent protein
Daizo Hamada1, Kouhei Tsumoto, Makoto Sawara
1Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. daizo@med.kobe-u.ac.jp
Abstract:
Green fluorescent protein (GFP) is often misfolded into nonfluorescent states when an aggregatable sequence is attached to its N-terminus. However, GFP fusions with highly aggregatable, prion-determining, and highly charged sequences from yeast prions, such as Sup35 and Ure2p, form green fibrils with properly folded GFP. To gain further insight into the general effect of an aggregatable sequence attached to fluorescent protein, we designed eight fusion proteins of a yellow variant of GFP (YFP) containing an aggregation-prone amyloidogenic sequence derived from human medin, attached via different lengths of linker sequence. Seven fusion proteins formed white fibrils lacking native YFP function. However, the fusion with an 18-residue medin sequence and a 50 amino acid linker formed fibrils with yellow color of folded YFP. Deconvolution analysis of infrared spectra also supports the presence of properly folded YFP in the fibrils formed by this protein. These results suggest that, the presence of an amyloidogenic sequence to a folded protein can promote the formation of fibrils and disrupt the native structures whereas the structure of the folded region is retained by optimizing sequences of amyloidogenic and linker regions.
Insights
Attaching aggregation-prone sequences to fluorescent proteins like yellow fluorescent protein (YFP) can create fibrils. Optimizing linker and sequence length can preserve YFP
Area of Science:
- Biochemistry
- Structural Biology
- Protein Aggregation
Background:
- Attaching aggregatable sequences to fluorescent proteins often leads to misfolding and loss of fluorescence.
- However, some yeast prion protein fusions (e.g., Sup35, Ure2p) with green fluorescent protein (GFP) form functional fluorescent fibrils.
- The general effect of aggregatable sequences on fluorescent protein structure and function remains incompletely understood.
Purpose of the Study:
- To investigate the impact of an aggregation-prone amyloidogenic sequence from human medin on yellow fluorescent protein (YFP) structure and fibril formation.
- To determine if optimizing linker length and sequence composition can maintain YFP's native structure and fluorescence within fibrils.
Main Methods:
- Design and construction of eight YFP fusion proteins with varying lengths of an 18-residue medin amyloidogenic sequence and linker regions.
- Characterization of fibril formation and YFP fluorescence in the engineered fusion proteins.
- Deconvolution analysis of infrared spectra to assess the structural integrity of YFP within the formed fibrils.
Main Results:
- Seven of the eight fusion proteins formed non-fluorescent white fibrils, indicating disruption of native YFP structure.
- One fusion protein, comprising the medin sequence and a 50 amino acid linker, formed yellow fibrils retaining YFP fluorescence.
- Infrared spectroscopy confirmed the presence of properly folded YFP within the fibrils of the optimized fusion construct.
Conclusions:
- Amyloidogenic sequences can induce fibril formation in fluorescent proteins, often disrupting their native structure.
- Careful optimization of the amyloidogenic sequence and linker region is crucial for retaining the folded structure and function of the attached protein.
- This study provides insights into designing functional protein-based amyloid fibrils.
