Effect of an amyloidogenic sequence attached to yellow fluorescent protein

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.

Hyperthin nanochains composed of self-polymerizing protein shackles

“…In a previous study in which amyloidogenic peptides tagged with yellow fluorescent protein were polymerized to form amyloid fibrils, most of the fluorescence was lost upon polymerization 46 , which suggests that the yellow fluorescent protein domains denature due to their ARTICLE dense packing. In our case, the GFP domains were positioned along the nanochain at B10-nm intervals, which allowed the GFP domains to retain their fluorescence after the GFP-PS was polymerized.…”

Towards Multiparametric Fluorescent Imaging of Amyloid Formation: Studies of a YFP Model of α-Synuclein Aggregation

“…In addition to the abovementioned inner filter effect, we note that absorption flattening (i.e., reduced absorption of photons by the chromophores due to shielding effects at high concentrations) is likely to be another major optical effect limiting the excitation of the YFP fluorophore in the aggregated sample. 49 Reduced fluorescence intensity could also result from the misfolding of a partial population of YFP molecules in the fibril state, as reported for some green fluorescent protein (GFP) chimeras of amyloidogenic peptides, 35 giving rise to a decrease in the extinction coefficient and quantum yield of the fluorophore. Absorption spectroscopy showed, however, that aggregated AS-YFP maintains the ratio of both chromophore/aromatics absorbance and YFP fluorescence/absorbance compared to monomeric AS-YFP, suggesting that misfolding of YFP is not a major cause for the decrease in brightness in this case ( Supplementary Fig.…”

“…35 To investigate the amyloid aggregation behavior of AS-YFP, we performed protein aggregation assays on the purified chimeric protein; as a control, wildtype AS was assayed in parallel. Both proteins were subjected to standard in vitro aggregation procedures, incubating the purified proteins at 37°C with constant agitation.…”

“…Indeed, many animal models of disease have been established, employing fluorescent protein tagging to study misfolding and accumulation of AS, 27-31 along with other misfolding disease-related proteins. [32][33][34][35] Conjugation of proteins by fluorescent proteins may, however, perturb the native structure and function of the tagged protein given the bulkiness of the fluorescent moiety (∼ 28 kDa). In the case of AS, this may be of particular concern, not only because of its relatively small size (∼ 14 kDa) but also because of its lack of persistent structure in solution.…”

Scanning Electron Microscope Imaging of Amyloid Fibrils

“…Amyloid fibrils are of considerable interest because amyloidogenesis has often been linked to fatal degenerative disorders, such as Alzheimer's and Parkinson's diseases. However, the well-defined structure and biocompatibility of amyloid fibrils have also suggested new functional materials (Knowles and Buehler, 2011), such as amyloid fibrils attached to yellow fluorescent protein (Hamada et al, 2008), insulin amyloids as a biomaterial for cell culture surfaces (Sakono et al, 2011) and templates of peptide fibres for metal nanowire (Reches and Gazit, 2003).…”