Abstract
Overexpression of recombinant proteins in bacteria may lead to their aggregation and deposition in inclusion bodies. Since the conformational properties of proteins in inclusion bodies exhibit many of the characteristics typical of amyloid fibrils. Based on these findings, we hypothesize that the rate at which proteins form amyloid fibrils may be predicted from their propensity to form inclusion bodies. To establish a method based on this concept, we first measured by SDS-PAGE and confocal microscopy the level of inclusion bodies in E. coli cells overexpressing the 40-residue amyloid-beta peptide, Aβ40, wild-type and 24 charge mutants. We then compared these results with a number of existing computational aggregation propensity predictors as well as the rates of aggregation measured in vitro for selected mutants. Our results show a strong correlation between the level of inclusion body formation and aggregation propensity, thus demonstrating the power of this approach and its value in identifying factors modulating aggregation kinetics.
Highlights
Alzheimer’s disease (AD) is associated with abnormalities in protein folding, resulting in the misfolding and aggregation of a characteristic set of proteins[1,2,3,4]
No expression in the soluble fraction could be detected by SDS-PAGE analysis, this is likely a combination of rapid formation of inclusion bodies by the aggregation-prone Aβ40 and because the peptide is relatively small and unstructured and gets degraded in E. coli if not protected in the form of inclusion bodies
All properties except β-sheet propensity give a positive contribution and charge has the most important contribution. These results show that the sign of the change in charge is the most important factor for determining the level of inclusion body formation, that it is dependent on other factors, and that the less negative and more negative mutants may be sensitive to different factors
Summary
Alzheimer’s disease (AD) is associated with abnormalities in protein folding, resulting in the misfolding and aggregation of a characteristic set of proteins[1,2,3,4]. A powerful approach to obtain such understanding is to perform the systematic kinetic analysis of a series of designed mutations of the Aβ sequence combined with changes in the solution composition[15,16,17] The results of such studies indicate that the aggregation of unstructured Aβ monomers into highly ordered amyloid fibrils is a complex process that is influenced by a vast number of factors. We use an Aβ40 mutant library of the wild-type protein and 24 single mutation variants (Fig. 1 and see Supplementary Table S1), in which we have changed all charged positions to oppositely charged or neutral hydrophilic amino acid residues, and all His and Asn residues to charged ones The rational for this design comes from earlier studies that indicated that electrostatic interactions strongly affect the aggregation kinetics of Aβ peptides[16,18,20,22,34]. Using a charge mutant library should provide a benchmark for an inclusion-body-based screening method
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