A direct sensor of O(2), the Dos protein, has been found in Escherichia coli. Previously, the only biological sensors known to respond to O(2) by direct and reversible binding were the FixL proteins of Rhizobia. A heme-binding region in Dos is 60% homologous to the O(2)-sensing PAS domain of the FixL protein, but the remainder of Dos does not resemble FixL. Specifically, the C-terminal domain of Dos, presumed to be a regulatory partner that couples to its heme-binding domain, is not a histidine kinase but more closely resembles a phosphodiesterase. The absorption spectra of Dos indicate that both axial positions of the heme iron are coordinated to side chains of the protein. Nevertheless, O(2) and CO bind to Dos with K(d) values of 13 and 10 microM, respectively, indicating a strong discrimination against CO binding. Association rate constants for binding of O(2) (3 mM(-)(1) s(-)(1)), CO (1 mM(-)(1) s(-)(1)) and even NO (2 mM(-)(1) s(-)(1)) are extraordinarily low and very similar. Displacement of an endogenous ligand, probably Met 95, from the heme iron in Dos triggers a conformational change that alters the activity of the enzymatic domain. This sensing mechanism differs from that of FixL but resembles that of the CO sensor CooA of Rhodospirillum rubrum. Overall the results provide evidence for a heme-binding subgroup of PAS-domain proteins whose working range, signaling mechanisms, and regulatory partners can vary considerably.

The M13 phage procoat protein requires both its signal sequence and its membrane anchor sequence in the mature part of the protein for membrane insertion. Translocation of its short acidic periplasmic loop is stimulated by the proton motive force (pmf) and does not require the Sec components. We now find that the pmf becomes increasingly important for the translocation of negatively charged residues within procoat when the hydrophobicity of the signal or membrane anchor is incrementally reduced. In contrast, we find that the pmf inhibits translocation of the periplasmic loop when it contains one or two positively charged residues. This inhibitory effect of the pmf is stronger when the hydrophobicity of the inserting procoat protein is compromised. No pmf effect is observed for translocation of an uncharged periplasmic loop even when the hydrophobicity is reduced. We also show that the ⌬⌿ component of the pmf is necessary and sufficient for insertion of representative constructs and that the translocation effects of charged residues are primarily due to the ⌬⌿ component of the pmf and not the pH component.All membrane proteins must adopt their correct asymmetric orientations in the membrane in order to function correctly. This is achieved according to two major known parameters. The hydrophobic stretches within membrane proteins enable the protein to partition into the membrane and span the bilayer, whereas the hydrophilic regions are retained at the correct face of the membrane as dictated by the "positive inside" rule (1). This rule states that regions rich in positively charged residues are directed to the cytoplasmic face of the inner membrane of Escherichia coli (2, 3). The basis for the positive inside rule has not been pursued diligently until recently (4, 5). One hypothesis is that the proton motive force, ⌬ H ϩ (pmf), 1 which renders the cytoplasm basic and the periplasm acidic, favors a cytoplasmic location for positively charged amino acids. The electrical component of the pmf, ⌬⌿, imposes an "uphill" barrier for positively charged residues to translocate into the periplasm, whereas negatively charged residues move downhill with the pmf (2). The pmf has been shown to drive the insertion of hydrophilic domains of both sec-dependent (6 -8) and sec-independent proteins (9 -11).Evidence for an electrophoretic membrane insertion mechanism has been reported for mutants of the exported protein proOmpA (12) and for derivatives of leader peptidase and procoat (4, 13). It was shown that the pmf promotes the translocation of negatively charged residues within a short periplasmic loop (4, 13) and an amino-terminal tail (13) of a membrane protein. In addition, the pmf appears to impede translocation of an amino-terminal tail when it possesses a positive charge (13) since the tail inserts more efficiently in the absence of the potential. The pmf also contributes to the topology of the protein, since abolishing the pmf causes a partial topology inversion in various positively charged mutants (4). However, similar stu...

No abstract

No abstract