Enzymes: Escherichia coli PBP4 (EC 188.8.131.52; EC 3.4.99-); E. coli PBP5 (EC 184.108.40.206); E. coli PBP6 (EC 220.127.116.11); E. coli PBP6b (EC 18.104.22.168).
D. Phoenix, Department of Forensic and Investigative Science, University of Central Lancashire, Preston, PR1 2HE, UK. Fax: + 44 1772 894964, Tel.: + 44 1772 893481, E-mail: firstname.lastname@example.org
Escherichia coli low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C-terminal amphiphilic α-helices. Nonetheless, the membrane interactive mechanisms utilized by the C-terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment-based analyses have predicted that, in contrast to the PBP4 and PBP6b C-termini, those of PBP5 and PBP6 are candidates to form oblique orientated α-helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C-terminal regions, P4 and P5, respectively, both possessed the ability to adopt α-helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal α-helices, the PBP5 and PBP6 C-terminal α-helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.
The Escherichia coli, low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 [1,2], PBP6b , PBP7 and PBP8 [4,5]. These proteins are penicillin sensitive DD-peptidases [6,7] that are believed to play a role in the final stages of petidoglycan manufacture [8–11]. PBP7 and PBP8 are soluble proteins [4,5] but it has been established that in nonoverproducing systems, PBP4, PBP5 and PBP6 are anchored to the periplasmic face of the inner membrane [7,12] whilst a similar membrane location has been suggested for PBP6b . Nonetheless, hydropathy plot analysis for each of these membrane-associated PBPs shows no conventional hydrophobic anchor sequences, nor did there appear to be any evidence of covalent modification and the membrane anchoring mechanisms of these proteins remained unclear. Deletion analysis showed that the C-terminal region of PBP5 [13,14] and PBP6  were essential for efficient membrane interaction whilst CD analysis showed that a peptide homolog of the PBP5 C-terminal region was able to adopt high levels of α-helical structure . Furthermore, incorporation of a proline residue into the PBP5 C-terminal region, with its ability to disrupt or distort α-helical structure, greatly destabilized the membrane anchoring of the protein  whilst fusion of the PBP5 C-terminal region to a periplasmic β-lactamase led to a membrane bound form of the enzyme .
A number of authors have used theoretical analysis to investigate the potential of the PBP4, PBP5, PBP6 and PBP6b C-terminal regions for membrane interaction and based on these analyses, it would appear that these C-terminal regions form two distinguishable subgroups. Both hydrophobic moment-based analyses [19,20] and DWIH analysis  have predicted that the PBP5 and PBP6 C-terminal regions would form strongly amphiphilic α-helices and in both cases, these predictions appear to be supported by experimental results, which found that peptide homologs of these regions were strongly hemolytic  and showed high levels of lipid monolayer penetration . In contrast, similar theoretical analyses have predicted that the PBP4 and PBP6b C-terminal regions would form weakly amphiphilic α-helices [19,21,24]. In the case of the PBP4 C-terminal region, these predictions could be supported by experimental results, which found that a peptide homolog of this region possessed no hemolytic ability  and low levels of lipid monolayer penetration . In addition, hydrophobic moment profile analysis has predicted that the PBP4 C-terminal region possesses an almost equal potential to interact with membranes via either amphiphilic β-sheet or amphiphilic α-helical secondary structures . Based on these results, it has been suggested that PBP4 and PBP6b may utilize mechanisms of membrane interaction, which differ to those utilized by PBP5 and PBP6 [1,2,19,21,24]. Here, we have considered the possibility that differences between these anchoring mechanisms may lay in the ability of the low molecular mass PBPs to form oblique orientated α-helices in their C-terminal regions. Recently confirmed by experimental results [25,26], these are a class of α-helices whose lipid interactions are predicted to involve penetration of the membrane at a shallow angle due to a hydrophobicity gradient along the α-helical long axis [27–30]. Using graphical and hydrophobic moment-based analyses, we have examined the PBP4, PBP5, PBP6 and PBP6b C-terminal sequences to identify candidate oblique orientated α-helix forming segments. In an effort to confirm this potential, we then used FTIR spectroscopy to investigate the ability of the PBP4 and PBP5 C-terminal sequences to adopt secondary structure at a lipid interface and for lipid interaction. Conformational analyses of peptide homologs of these sequences, P4 and P5, respectively, were performed in the presence of vesicles formed from either: Myr2-PGro, Myr2-PCho or Myr2-PEtn. FTIR spectroscopy was then used to monitor the effects of P4 and P5 on the phase transition temperature and membrane fluidity of membranes formed from either: Myr2-PGro, Myr2-PCho or Myr2-PEtn.
Materials and methods
The identification of candidate oblique orientated α-helix forming segments
The primary sequence of the influenza viral fusion peptide, HA2, a known oblique orientated α-helix former [31–33] and those of the PBP4, PBP5, PBP6 and PBP6b C-terminal regions (Table 1) were analyzed according to conventional hydrophobic moment methodology . The hydrophobicity of successive amino acids in these sequences are treated as vectors and summed in two dimensions, assuming an amino acid side chain periodicity of 100°. The resultant of this summation, the hydrophobic moment, µH, provides a measure of α-helix amphiphilicity. Our analysis used a moving window of 11 residues and for each sequence under investigation (Table 1), the window with the highest hydrophobic moment was identified (Table 1). For these windows, the mean hydrophobic moment, 〈µH〉, and the corresponding mean hydrophobicity, 〈H0〉 (Table 1), were computed using the normalized consensus hydrophobicity scale of Eisenberg et al. and plotted on the hydrophobic moment plot diagram , as modified by Harris et al.(Fig. 1).
Table 1. The primary sequences and hydrophobic moment parameters of protein segments. The C-terminal sequences of PBP4 , PBP5 and PBP6 , PBP6b  and the primary sequence of the HA2 fusion peptide  were analyzed using hydrophobic moment methodology . Eleven residue windows of maximum amphiphilicity were identified (shown in bold) and are shown, along with their corresponding 〈µH〉 and 〈H0〉.
++ + − ++− −
+ − +++ −
+ − +++ −
− − + − +++ −
− − −
HA2 fusion peptide
The peptides P4 and P5 (Table 1) were supplied by PEPSYN, University of Liverpool, UK, produced by solid state synthesis and purified by HPLC to a purity of greater than 99%. The peptides were stored as 1 m aqueous stock solutions at 4 °C. Myr2-PGro, Myr2-PCho and Myr2-PEtn, and all solvents, which were of spectroscopic grade, were purchased from Sigma (UK).
Preparation of phospholipid small unilamellar vesicles
Small unilamellar vesicles (SUVs) were prepared according to Keller et al.. Essentially, lipid/chloroform mixtures were dried with nitrogen gas and hydrated with aqueous Hepes at pH 7.5 to give final phospholipid concentrations of 50 mm. The resulting cloudy suspensions were sonicated at 4 °C with a Soniprep 150 sonicator (amplitude 10 µm) until clear suspensions resulted (30 cycles of 30 s), which were then centrifuged (15 min, 3000 g, 4 °C).
FTIR conformational analyses of P4 and P5
To give a final peptide concentration of 1 mm, either P4 or P5 were solubilized in either aqueous buffer (50 mm Hepes; pH 7) or suspensions of SUVs, which were formed from either: Myr2-PGro, Myr2-PCho or Myr2-PEtn, and were prepared as described above. Samples of solubilized peptide were spread on a CaF2 crystal, and the free excess water was evaporated at room temperature. The single band components of the P4 or P5 amide I vibrational band (predominantly C=O stretch) was monitored using an FTIR ‘5-DX’ spectrometer (Nicolet Instruments, Madison, WI, USA).
Analysis of FTIR spectra
FTIR spectra were analyzed and for those with strong absorption bands, the evaluation of the band parameters (peak position, band width and intensity) was performed with the original spectra, if necessary after the subtraction of strong water bands. In the case of spectra with weak absorption bands, resolution enhancement techniques such as Fourier self-deconvolution  were applied after baseline subtraction with the parameters: bandwidth, 22–28 cm−1, resolution enhancement factor, 1.2–1.4 and Gauss/Lorentz ratio of 0.55. In the case of overlapping bands, curve fitting was applied using a modified version of the curfit procedure written by D. Moffat (National Research Council, Ottowa, Canada). An estimation of the number of band components was obtained from deconvolution of the spectra, curve fitting was then applied within the original spectra after the subtraction of baselines resulting from neighboring bands. Similar to the deconvolution technique, the bandshapes of the single components are superpositions of Gaussian and Lorentzian bandshapes. Best fits were obtained by assuming a Gauss fraction of 0.55–0.6. The curfit procedure measures the peak areas of single band components and after statistical evaluation, determines the relative percentages of primary structure involved in secondary structure formation. For P4 and P5, relative levels of α-helical structure (1650–1655 cm−1) and β-sheet structures (1625–1640 cm−1) were computed and are shown in Table 2.
Table 2. . P4 and P5 secondary structural levels in the presence of lipid. Levels of secondary structure determined for P4 and P5. FTIR conformational analysis of P4 and P5 were performed with each peptide either: in aqueous solution (–) or in the presence of either: dimyristoyl phosphatidylcholine (Myr2-PCho), dimyristoyl phosphatidylethanolamine (Myr2-PEtn), or dimyristoyl phosphatidylglycerol (Myr2-PGro). For spectra produced (Figs 2 and 5), the peak areas of single band components were used to determine the relative percentages of primary structure involved in secondary structure formation.
α-helical structures (%)
β-sheet structures (%)
FTIR analysis of phospholipid phase transition properties
Using FTIR spectroscopy, the effects of either P4 or P5 on the phase transition properties of phospholipid was investigated. To give a final peptide concentration of 1 mm, either P4 or P5 was solubilized in suspensions of SUVs formed from: either Myr2-PGro, Myr2-PCho or Myr2-PEtn, which were prepared as described above. As controls, SUVs formed from: either Myr2-PGro, Myr2-PCho or Myr2-PEtn alone were prepared as described above. These samples were then placed in a calcium fluoride cuvette, separated by a 12.5-µm thick Teflon spacer and subjected to automatic temperature scans with a heating rate of 3 °C 5 min−1 within the temperature range 0 to 60 °C. For every 3 °C interval, 50 interferograms were accumulated, apodized, Fourier transformed and converted to absorbance/ temperature spectra  (Figs 3 and 6). These spectra monitored changes in the β→α acyl chain melting behavior of phospholipids with these changes determined as shifts in the peak position of the symmetric stretching vibration of the methylene groups, νs(CH2), which is known to be a sensitive marker of lipid order. The peak position of νs(CH2) lies at 2850 cm−1 in the gel phase and shifts at a lipid specific temperature Tc to 2852.0 cm−1−2852.5 cm−1 in the liquid crystalline state.
The identification of candidate oblique orientated α-helix forming segments
The sequences shown in Table 1 were plotted on the modified hydrophobic moment plot diagram (Fig. 1) according to their 〈µH〉 and 〈H0〉 values (Table 1). Data points representing the PBP5 and PBP6 C-terminal sequences are seen to lay within the shaded area, proximal to that representing the sequence of HA2, a known oblique orientated α-helix former. These observations indicate that the PBP5 and PBP6 C-terminal sequences are candidate oblique orientated α-helix forming segments. However, data points representing the PBP4 and PBP6b C-terminal regions are seen to lay outside the shaded area, indicating that these sequences are unlikely to form oblique orientated α-helices (P > 0.01 confidence).
FTIR conformational analysis of peptides
FTIR spectroscopy was used to perform conformational analyses of P4 and P5 either in aqueous solution or in the presence of SUVs. A typical overview spectrum for these peptide–lipid systems is shown in Fig. 4, which represents absorbance by the P4-Myr2-PCho system within the spectral range 1800–1100 cm−1. The spectrum comprises lipid vibrational bands such as the ester double bond stretching at 1738 cm−1, the methylene chain scissoring mode at 1464 cm−1 , and the phosphate antisymmetric stretching at 1240–1200 cm−1, and the peptide bands, amide I (predominantly C=O stretching) and amide II (predominantly N–H bending). Figures 2 and 5 show spectra for P4 and P5 absorbance in the spectral range of the amide I band and from these spectra, the relative levels of peptide secondary structure were determined as a percentage of primary structure (Table 2). The major contribution to P4 molecular architecture came from β-sheet structures, ranging from 63% in the presence of Myr2-PEtn to 85% in the aqueous peptide. Nonetheless, the peptide adopted significant levels of α-helical structure in the presence of both Myr2-PEtn (37%) and Myr2-PGro (20%) although showing no evidence of such structure either in the presence of Myr2-PCho or in aqueous solution (Table 2). In contrast, P5 showed high levels of α-helical structure in aqueous solution (58%), which were generally maintained in the presence of Myr2-PCho, Myr2-PEtn and Myr2-PGro and ranged between 43% and 56%.
The effect of proteins on phospholipid phase transition temperature
Using FTIR spectroscopy, absorbance spectra representing the effects of either P4 or P5 on the phase transition temperature and membrane fluidity of membranes formed from either: Myr2-PCho, Myr2-PGro or Myr2-PEtn, were derived as a function of temperature (Figs 3 and 6). Control experiments recorded the Tc of both Myr2-PGro and Myr2-PCho membranes as 25 °C and that of Myr2-PEtn membranes as 47 °C (Figs 3A–C and 6A−C). In the presence of P4, no significant changes in either the membrane fluidity or the Tc of Myr2-PCho membranes were detected (Fig. 3A). Similarly, the presence of P4 appeared to have no significant effect on Myr2-PEtn membrane fluidity but did appear to have a significant effect on the Tc of the lipid, with Tc being recorded as 42 °C in the presence of the peptide (Fig. 3B). The presence of P5 had a strong effect on the Tc and membrane fluidity of both Myr2-PCho and Myr2-PEtn membranes with Tc being recorded as 13 and 42 °C, respectively, and in each case, the change was accompanied by a concomitant increase in membrane fluidity (Fig. 6A,B). In contrast, in the presence of either P4 or P5, Myr2-PGro membranes showed minor increases in gel phase fluidity, minor decreases in liquid crystalline phase fluidity with the gel to fluid phase transition occurring over the interval 20 to 30 °C rather than the 25 °C of the pure lipid (Figs 3C and 6C).
Here, we analyzed the C-terminal sequences of PBP4, PBP5, PBP6 and PBP6b to identify candidates with the potential to form oblique orientated α-helices [27,30] and based on their 〈µH〉 , and 〈H0〉 values, our analyses showed that these sequence formed two subgroups. The C-terminal regions of PBP4 and PBP6b were predicted to be unlikely to form oblique orientated α-helices. However, the C-terminal regions of PBP5 and PBP6 were predicted to be candidates for the formation of such α-helices and are similar to the viral fusion peptide, HA2 (Fig. 1), a peptide shown to penetrate membranes via an oblique orientated α-helix. The C-terminal α-helices of PBP5 and PBP6 show many structural resemblances to the HA2 oblique orientated α-helix. It can be seen from Fig. 7 that each of these α-helices possesses a wide hydrophobic face, which includes bulky tryptophan, phenylaniline and isoleucine amino acid residues, and a glycine rich hydrophilic face. These structural features give α-helices an effective wedge shape, which appears to be utilized by HA2, and a number of other oblique orientated α-helix forming peptides, to destabilize membranes, leading to membrane fusion [40,41]. Furthermore, Roberts et al. analyzed the PBP5 and PBP6 C-terminal α-helices according to DWIH methodology and showed that the nature and order of the amino acid residues forming these α-helices were highly significant. This is consistent with the ordered spatial arrangement of amino acid residues necessary to maintain the hydrophobicity gradients of oblique orientated α-helices . In contrast to the PBP5 and PBP6 C-terminal regions, it can be seen from Fig. 7 that, in an α-helical conformation, the C-terminal regions of PBP4 and PBP6b show ill-defined hydrophobic faces and few structural resemblances to the HA2 α-helix. These observations reinforce the suggestion that there would be differences between the C-terminal membrane interactions for PBPs from the two subgroups.
The PBP4 and PBP5 C-terminal anchor sequences were taken to represent each of these subgroups and the secondary structural features of these sequences in the presence of lipid have been investigated. FTIR conformational analysis showed that both in aqueous solution and in the presence of each lipid examined, over 60% of P4 architecture was formed from β-sheet structures (Fig. 5A–D; Table 2). Nonetheless, in the presence of both Myr2-PEtn and Myr2-PGro (Fig. 5B,C; Table 2), the peptide adopted significant levels of α-helical structure (37% and 20%, respectively) although showing no evidence of such structure either in the presence of Myr2-PCho (Fig. 5A; Table 2) or in aqueous solution (Fig. 5D; Table 2). In contrast, P5 architecture showed high levels of α-helical structure, of the order of 50%, both in aqueous solution (Fig. 2D; Table 2) and in the presence of each lipid examined (Fig. 2A–C; Table 2). Both P4 and P5 were found to affect the lipid phase transition properties of Myr2-PEtn (Figs 3B and 6B) and Myr2-PGro (Figs 3C and 6C). However, whilst P5 was found to affect the lipid phase transition properties of Myr2-PCho (Fig. 6A) no such effects were detected in the case of P4 (Fig. 3A). In combination, these results would support the hypothesis that the ability of P4 and P5 to interact with lipid membranes is related to the ability of each peptide to adopt amphiphilic α-helical structure. Furthermore, these results are consistent with those of Brandenburg et al. and suggest that the ability of P4 to adopt such structure may be related to the characteristics of the interface rather than solely the lipid type.
Our FTIR lipid phase transition analyses showed that the presence of both P4 and P5 led to a broadening of the Tc of Myr2-PGro membranes (25 °C) with phase transition occurring over a temperature range (20–30 °C) accompanied by an increases in gel phase fluidity and decreases in liquid crystalline phase fluidity (Figs 3C and 6C), This form of phase transition shows similarities to that of some cholesterol–lipid systems  and implies that the presence of either P4 or P5 leads to changes in the hydrocarbon chain packing of Myr2-PGro membrane, which result in fluidization of the gel phase and rigidification of the liquid crystalline phase. These results do not necessarily mean that P4 and P5 interact with the Myr2-PGro acyl chains region and in isolation, do not allow a clear interpretation as to the nature of P4 and P5 interaction with Myr2-PGro membranes. Even so, these results clearly show that there is some level of Myr2-PGro membrane penetration by the peptides. The presence of P4 had no effect on the lipid phase transition properties of Myr2-PCho (Fig. 3A) and no effect on the membrane fluidity of Myr2-PEtn membranes, although a 5 °C decrease in the Tc of Myr2-PEtn was observed (Fig. 3B). P5 was found to interact strongly with Myr2-PCho membranes (Fig. 6A) and Myr2-PEtn membranes (Fig. 6B) with the presence of the peptide leading to increased membrane fluidity in both cases, accompanied by decreases in membrane Tc of 12 and 5 °C, respectively. Taken overall, these results clearly show that there are fundamental differences between the mechanisms of membrane penetration utilized by P4 and P5. P4 shows limited levels of membrane penetration and would appear to prefer to interact with the membrane's surface regions whilst P5 has a preference to interact with the membrane's lipid acyl chains.
Taken in combination, our experimental results are consistent with our suggestion that the PBP5 and PBP6 C-terminal α-helices may be able to penetrate the membrane lipid core region in an oblique orientation. This form of membrane penetration would be in accord with the high levels of interaction shown here by P5 for zwitterionic membranes. Furthermore, this form of membrane penetration could explain the high levels of hemolysis shown by both this peptide and P6, a peptide homolog of the PBP6 C-terminal region  for HA2 is hemolytic yet abolition of the peptide's hydrophobicity gradient leads to loss of hemolytic and fusogenic ability . Given the apparent preference shown by P4 for the membrane's surface regions, this suggests that the peptide's cationic region(s) would interact with negatively charged moieties within this region. Nonetheless, taking our results overall, we speculate that such an interaction would be weak and unlikely to play a major role in the membrane anchoring mechanism of PBP4. Indeed, experimental evidence has been presented which suggests that the membrane interactions of PBP4 may involve occupation of a specific binding site  and protein–protein interactions [8–11].
In summary, our results show that the PBP4 C-terminal sequence is able to adopt α-helical and β-sheet structure in the presence of lipid and may weakly associate with the membrane lipid headgroup region via predominantly electrostatic interactions. In contrast, our results suggest that the PBP5 C-terminal region possesses a strong intrinsic tendency to both adopt α-helical structure and to penetrate the membrane hydrophobic core region. It appears that this C-terminal α-helix, and that formed by PBP6, possess hydrophobicity gradients, which we suggest may facilitate membrane penetration in an oblique orientation.