Mutational analysis of the aggregation-prone and disaggregation-prone regions of acylphosphatase.
We have performed an extensive mutational analysis of aggregation and disaggregation of amyloid-like protofibrils of human muscle acylphosphatase. Our findings indicate that the regions that promote aggregation in 25% (v/v) 2,2,2 trifluoroethanol (TFE) are different from those that promote disaggregation under milder conditions (5% TFE). Significant changes in the rate of disaggregation of protofibrils in 5% TFE result not only from mutations situated in the regions of the sequence that play a key role in the mechanism of aggregation in 25% TFE, but also from mutations located in other regions. In order to rationalise these results, we have used a modified version of the Zyggregator aggregation propensity prediction algorithm to take into account structural rearrangements of the protofibrils that may be induced by changes in solution conditions. Our results suggest that a wider range of residues contributes to the stability of the aggregates in addition to those that play an important kinetic role in the aggregation process. The mutational approach described here is capable of providing residue-specific information on the structure and dynamics of amyloid protofibrils under conditions close to physiological and should be widely applicable to other systems.
Calamai
Martino
M
Department of Chemistry, University of Cambridge, Cambridge, UK.
Tartaglia
Gian Gaetano
GG
Vendruscolo
Michele
M
Chiti
Fabrizio
F
Dobson
Christopher M
CM
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics*; D000465:Algorithms; D019943:Amino Acid Substitution; D000682:Amyloid / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D000682:Amyloid; D011994:Recombinant Proteins; D014270:Trifluoroethanol; D000097024:Acylphosphatase