Two genetically engineered variants of the Bacillus licheniformis b-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 DG 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 b-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.Keywords: b-lactamase; protein export; protein folding; signal peptide.The information to guide protein folding and export is encoded by the amino acid sequence. As it is generally accepted that native structures do not cross membranes, it follows that protein folding must be coordinately regulated to allow protein export [1±4]. An additional complication is the potential for folding proteins to undergo aggregation, which could interfere with the kinetics of both folding and export.In some cases, folding is impeded cotranslationally, by attaching the functional ribosome to a membrane, sequestering the nascent peptide from the cytoplasm, and extruding it through a proteinaceous pore. In other cases, a much more interesting mechanism takes place that keeps a completely synthesized polypeptide in a non-native, water-soluble, exportcompetent state until translocation is achieved. In this mechanism, the folding code must be temporarily overridden by the export code.Most exported proteins have a signal sequence whose proposed functions are: (a) to keep the precursor in an export-competent state; (b) to interact with specific components of the export machinery; and (c) to initiate protein secretion by inserting itself in the membrane bilayer [5]. After proper localization, the signal is usually removed by specific proteases, releas...
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