Investigating the effects of mutations on protein aggregation in the cell.
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.
Calloni
Giulia
G
Dipartimento di Scienze Biochimiche, Università Degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
Zoffoli
Sara
S
Stefani
Massimo
M
Dobson
Christopher M
CM
Chiti
Fabrizio
F
D000818:Animals; D052160:Benzothiazoles; D001709:Biotechnology; D015153:Blotting, Western; D002645:Chickens; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012995:Solubility; D013844:Thiazoles / Q000737:chemistry*; D014508:Urea / Q000737:chemistry / Q000494:pharmacology
D052160:Benzothiazoles; D046911:Macromolecular Substances; D010455:Peptides; D013844:Thiazoles; C009462:thioflavin T; D014508:Urea; D009113:Muramidase