Trp replacements for conserved Gly-Gly pairs between the N- and C-terminal six-helix bundles on the periplasmic side of lactose permease (LacY) cause complete loss of transport activity with little or no effect on sugar binding. Moreover, the detergent-solubilized mutants exhibit much greater thermal stability than WT LacY. A Cys replacement for Asn245, which is inaccessible/unreactive in WT La...

Single chain Fragment variable (scFv) is an antibody fragment consisting variable regions of heavy and light chains. scFvs enhance their penetrability into tissues while maintaining specific affinity and having low immunogenicity. Insoluble inclusion bodies are formed when scFvs are expressed in reducing bacterial cytoplasm. One strategy for obtaining functionally active scFv is to translocate ...

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) dom...

Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the externa...

DegS, the periplasmic stress sensor, becomes activated when its PDZ domain recognizes the improperly exposed C-terminal sequences of outer membrane porins. This interaction relieves the inhibition of the neighboring protease domain of DegS, triggering a proteolysis cascade that leads to the sigma(E)-driven expression of periplasmic chaperones.

No periplasmic binding protein has been demonstrated for the ATP-binding cassette (ABC)-type cobalamin transporter BtuCD. New mutations (btuF) are described that affect inner-membrane transport. The BtuF protein has a signal sequence and resembles the periplasmic binding proteins of several other ABC transporters.

The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a beta-barrel protein, accompanied by translocatio...

The aim of this investigation was to compare the efficacy of the permeabilization methods of microwave irradiation and potassium phosphate (KPi)-hexane solvent system in isolating the periplasmic enzyme, L-asparaginase II. Previous studies have shown that both methods enable the isolation of periplasmic proteins in Gram-negative bacteria. Periplasmic, cytoplasmic and membrane fractions of Esche...

The lactose permease (LacY) catalyzes galactoside/H(+) symport via an alternating access mechanism in which sugar- and H(+)-binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by opening and closing of inward- and outward-facing cavities. The crystal structures of wild-type LacY, as well as accessibility data for the protein in the membrane, prov...

Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize peripla...