Investigating the Effects of Mutations on Protein Aggregation in the Cell

Abstract

The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.

Charge substitution shows that repulsive electrostatic interactions impede the oligomerization of Alzheimer amyloid peptides

“…Moreover, for a series of Ribonuclease Sa variants carrying charge-reversal mutations, the ability to remain soluble as a function of pH was found to be strictly correlated with net charge [34]. A high net charge has been identified as an important factor in preventing protein aggregation in vitro [35] and in vivo [36]. …”

“…The first 16 residues of Ab are rich in charged residues and the rest of the sequence contains two stretches of hydrophobic residues (17)(18)(19)(20)(21) and (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), believed to be key for oligomerization, separated by a region containing two acidic residues (Glu 22 and Asp 23) and a basic one (Lys 28) ( Table 1). Ab oligomerization is highly pH dependent.…”

Thioflavin-S Staining of Bacterial Inclusion Bodies for the Fast, Simple, and Inexpensive Screening of Amyloid Aggregation Inhibitors

“…Interestingly, bacterial systems can be easily implemented to study the effect of genetic mutations [52][53][54][55] and/or the effects of drugs [56] on the aggregation of amyloid-prone proteins. We present here a protocol in which the direct determination of the Th-S relative fluorescence of bacterial cells allows to monitor changes in the intracellular aggregation of amyloidogenic proteins, without the requirement for expensive equipment.…”

Amyloidogenic sequences in native protein structures

“…11,46,47 Similarly, in vivo experiments have shown that mutations decreasing the positive charge on a molecule increase the aggregation propensity and mutations increasing the net charge result in decreased aggregation. 48 Amyloidogenic proteins which are predominantly a-helical, must undergo an a-helix to b-sheet conversion during the formation of amyloid fibrils. 27 The tendency to form a helical secondary structure thus mitigates against the tendency to form b-sheet and hence amyloid fibrils.…”