Dimorphic aggregation behavior of a fusion polypeptide incorporating a stable protein domain (EGFP) with an amyloidogenic sequence (retroCspA).
We describe the behavior of a polypeptide consisting of the genetic fusion of a structurally stable single-domain protein, EGFP (an analog of the green fluorescent protein) with an amyloidogenic sequence, retroCspA (known to readily form amyloid fibrils). Refolding of the fusion protein through single-step removal of denaturant and salt results in precipitation into amyloid aggregates displaying fibrillar morphology, thioflavin T binding as well as green fluorescence. Refolding through step-wise reduction of denaturant concentration in the presence of salt yields a soluble aggregate containing a folded, thermally-stable, non-fluorescent EGFP domain. Together, these results indicate that retroCspA forces the fusion protein to aggregate; however, the EGFP domain remains folded in a native-like structural format in both soluble aggregates and precipitates.
Sharma
Swati
S
Protein Science and Engineering Division, Institute of Microbial Technology (IMTECH), Chandigarh, India.
Guptasarma
Purnananda
P
D000682:Amyloid / Q000737:chemistry*; D052160:Benzothiazoles; D057773:Cold Shock Proteins and Peptides; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008970:Molecular Weight; D014176:Protein Biosynthesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013844:Thiazoles / Q000737:chemistry
D000682:Amyloid; D052160:Benzothiazoles; D057773:Cold Shock Proteins and Peptides; C498192:CspA protein, E coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C009462:thioflavin T