Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli.
Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.
Bourot
S
S
Groupe Membranes et Osmorégulation, CNRS UPRES-A 6026, Université de Rennes I, Campus de Beaulieu, 35042 Rennes, France.
Sire
O
O
Trautwetter
A
A
Touzé
T
T
Wu
L F
LF
Blanco
C
C
Bernard
T
T
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001622:Betaine / Q000378:metabolism* / Q000494:pharmacology*; D001692:Biological Transport; D002262:Carboxy-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003470:Culture Media; D003960:Diaminopimelic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D005838:Genotype; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D001622:Betaine; D003960:Diaminopimelic Acid; D002262:Carboxy-Lyases; C079912:LysA protein, Bacteria; C489812:LysA protein, E coli; C020031:diaminopimelic acid decarboxylase