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Biomedical subjects

D A Phoenix

Publications and source records attributed to D A Phoenix.

53 records · Page 3Linked to original sources
PubMed1994

Analysis of the membrane-anchoring properties of the putative amphiphilic alpha-helical anchor at the C-terminus of Escherichia coli PBP 6.

Penicillin-binding protein (PBP) 6 is anchored to the periplasmic face of the Escherichia coli inner membrane. Analysis of the C-terminal 20 amino acids of PBP 6 implies the presence of a C-terminal amphiphilic alpha-helical anchor comparable to that of PBP 5. A C-terminal deletion of PBP 6 was constructed; it resulted in the release of the protein from the inner membrane into the periplasm, thus confirming that this region is essential for anchoring. Treatment of E. coli K12 membrane vesicles with various reagents was used to probe the membrane-binding characteristics of both PBP 5 and PBP 6. The results indicate that, although the strength of membrane anchoring of PBP 6 is weaker than that of PBP 5, both modes of anchoring involve a large hydrophobic element and have similar membrane-binding characteristics. This is in agreement with the hypothesis that both proteins exhibit the same novel method of anchoring.

Amino Acid Sequence↗
PubMed1994

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins.

The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M(r) PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M(r) PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and 6 are DD-carboxypeptidases. All three low M(r) PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic alpha-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.

Amino Acid Sequence↗
PubMed1993

OmpF-Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol-signal sequence interaction during protein translocation across the Escherichia coli inner membrane.

Using inverted Escherichia coli inner membrane vesicles we have analyzed the phosphatidylglycerol dependence of translocation of an OmpF-Lpp fusion protein carrying a signal sequence with varying positive charge at the N terminus and a hydrophobic core of varying length. It is shown that there is a direct relationship between the phosphatidylglycerol requirement of translocation and the requirement within the translocation process for positive charges on the signal sequence. This provides further evidence that the negative head group of the lipid is required for functional interaction with the positively charged N terminus of the signal sequence.

Bacterial Outer Membrane Proteins↗
PubMed1993

Phosphatidylglycerol dependent protein translocation across the Escherichia coli inner membrane is inhibited by the anti-cancer drug doxorubicin. Evidence for an electrostatic interaction between the signal sequence and phosphatidylglycerol.

OmpF-Lpp, a model secretory protein, requires both a positively charged signal sequence and phosphatidylglycerol (PG) for efficient translocation across the E. coli inner membrane. Modification of the signal sequence can, however, remove both these prerequisites for translocation providing OmpF-Lpp mutants which undergo either PG and charge dependent or PG and charge independent translocation. Here we show that positively charged membrane interactive compounds (polylysine & doxorubicin) are able to inhibit PG dependent translocation of the OmpF-Lpp signal sequence mutants but not PG independent translocation. Doxorubicin is also shown to bind more efficiently to liposomes containing increased levels of anionic lipid indicating that in these assays it may be inhibiting translocation by preventing electrostatic interaction between the anionic lipid head group and the positively charged signal sequences.

Bacterial Outer Membrane Proteins↗
PubMed1993

Membrane interaction of Escherichia coli penicillin binding protein 5 is modulated by the ectomembranous domain.

E. coli penicillin binding protein (PBP) 5 is anchored to the periplasmic face of the inner membrane by a C-terminal domain which is predicted to form an amphiphilic alpha-helix. Here we show that the presence of a substrate analogue, benzyl penicillin, causes the protein to be converted from a membrane bound urea inaccessible form to a urea extractable form. If the anchor region is fused to the periplasmic protein, beta-lactamase, the fusion protein becomes membrane bound but is unable to exhibit the changes in urea extractability which are observed with PBP5. We therefore conclude that although the C-terminus of PBP5 is sufficient to anchor the protein to the membrane surface the ectomembranous domain can affect the state of the anchor and in vivo changes in the state of anchoring may be related to enzyme activity.

Bacterial Proteins↗
PubMed1990

pH-induced insertion of the amphiphilic alpha-helical anchor of Escherichia coli penicillin-binding protein 5.

By treating vesicles prepared from Escherichia coli K12 with various reagents, we have investigated the mechanism by which penicillin-binding protein 5 anchors to the inner membrane. The results indicate that there are two forms of anchoring; one which is inaccessible to urea and probably inserted into the bilayer and one which is accessible. Association of the accessible form with the membrane seems to involve significant hydrophobic interaction and this form is triggered to undergo reversible 'insertion' by a decrease in pH.

Bacterial Proteins↗
PubMedDate not supplied

Use of hydrophobic moment plot methodology to aid the identification of oblique orientated alpha-helices.

A number of alpha-helix forming peptides have been reported which appear to promote membrane fusion and other biological events related to the disruption of a hydrophobic/hydrophilic interface, due to the presence of a hydrophobicity gradient along the helical long axis. When alpha-helices from this class were analysed according to hydrophobic moment plot methodology a linear association was found to exist between the mean hydrophobic moment, , and the corresponding mean hydrophobicity, . This association was described by the least squares regression line: =0.508-0.422 and, here, a methodology to aid the prediction of oblique orientated alpha-helices is presented, based on a 99% prediction band around this regression line. This methodology is intended to provide an initial identification of candidates for further investigation by other techniques such as the molecular hydrophobic potential and laboratory based experimentation, not to assign function.

Amino Acid Sequence↗