Influence of the stability of a fused protein and its distance to the amyloidogenic segment on fibril formation

• Published in: PloS one 2010-11, Vol.5 (11), p.e15436-e15436
• Main Authors: Buttstedt, Anja, Winter, Reno, Sackewitz, Mirko, Hause, Gerd, Schmid, Franz-Xaver, Schwarz, Elisabeth
• Format: Article
• Language: English
• Subjects: Alanine; Alanine - chemistry; Alanine - genetics; Alanine - metabolism; Amino acids; Amyloid; Amyloid - chemistry; Amyloid - genetics; Amyloid - metabolism; Amyloidogenesis; Analysis; Bacillus subtilis; Bacillus subtilis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Biochemistry; Biology; Biotechnology; Circular Dichroism; Cold; Control stability; Deoxyribonucleic acid; Disease; DNA; Fibrillation; Fibrils; Fused protein; Gene expression; Mutagenesis; Mutation; Oligopeptides - chemistry; Oligopeptides - genetics; Oligopeptides - metabolism; Poly(A)-Binding Proteins - genetics; Proteases; Protein Binding; Protein folding; Protein Stability; Protein Unfolding - drug effects; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Spectrometry, Fluorescence; Stability analysis; Structural stability; Thermodynamics; Urea - chemistry; Urea - pharmacology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0015436

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔG(D) = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔG(D) = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.