Creation of a Fully Functional Cysteine-Less Variant of Osmosensor and Proton-Osmoprotectant Symporter ProP from Escherichia coli and Its Application to Assess the Transporter's Membrane Orientation

Abstract

Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parameters describing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His)(6) variants were examined, including the threshold for osmotic activation and the absolute transporter activity at high osmolality (in both cells and proteoliposomes), the dependence of K(M) and V(max) for proline uptake on osmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cells only). Variant ProP-(His)(6)-C112A-C133A-C264V-C367A (designated ProP) retained similar activities to ProP-(His)(6) in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser at position 264, whereas Val strongly impaired function when placed at position 367, highlighting the importance of residues at those positions for osmosensing. In the ProP* background, variants with a single Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys at positions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorly reactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 were consistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmic membrane, respectively. These observations are consistent with the topology and orientation of ProP predicted by hydropathy analysis.

Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP, LacY, ProW, AqpZ, MscS, and MscL

“…Bacteria were cultivated as described previously for transport assays (12) in low-osmolality MOPS medium supplemented with arabinose (1 mM), and SDS-PAGE was performed as described by Laemmli (26) using Bio-Rad MiniProtean II cells (Bio-Rad Laboratories Ltd., Hercules, CA) (12% T, 2.67% C). The SDS loading buffer did not contain ␤-mercaptoethanol.…”

“…For Western blotting, bacteria were cultivated as described previously for transport assays (12) in low-osmolality MOPS medium without arabinose. Cell extracts were separated by SDS-PAGE, and Western blotting was performed using anti-ProP antibodies as described previously (49).…”

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

“…The osmolality at which ProP activity is half-maximal (⌸ 1/2 /RT) depends on the structure of the transporter (21,27,28,34,48) and the solvent to which it is exposed (42). The positive correlations among growth medium osmolality, the CL content of E. coli, and ⌸ 1/2 /RT for ProP suggested that ⌸ 1/2 /RT is also elevated by a CL-rich membrane environment (7).…”

Use of EPR Power Saturation to Analyze the Membrane-Docking Geometries of Peripheral Proteins: Applications to C2 Domains

“…When native cysteines are present, however, a cysteine-less version of the protein can be constructed by converting each cysteine to alanine or serine. Previous studies have indicated that such engineered cysteine-less proteins typically retain native structure and function (10,25,37,40). Native cysteines that are protected from labeling by a highly buried location, by involvement in a disulfide bond, or by covalent modification such as myristoylation do not interfere with site-directed spin labeling and can be retained.…”