Export and folding of signal-sequenceless Bacillus licheniformis beta-lactamase in Escherichia coli.
Two genetically engineered variants of the Bacillus licheniformis beta-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DeltaG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli beta-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.
Frate
M C
MC
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina.
Lietz
E J
EJ
Santos
J
J
Rossi
J P
JP
Fink
A L
AL
Ermácora
M R
MR
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001692:Biological Transport; D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D019897:Periplasm / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D001618:beta-Lactamases