Báo cáo Y học: Investigations into the mechanisms used by the C-terminal anchors of Escherichia coli penicillin-binding proteins 4, 5, 6 and 6b for membrane interaction ppt

Investigations into the mechanisms used by the C-terminal anchorsfor membrane interaction Frederick Harris1, Klaus Brandenburg2, Ulrich Seydel2and David Phoenix1 1 Department of Forensic

Investigations into the mechanisms used by the C-terminal anchors

for membrane interaction

Frederick Harris1, Klaus Brandenburg2, Ulrich Seydel2and David Phoenix1

1

Department of Forensic and Investigative Science, University of Central Lancashire, Preston, UK;2Division of Biophysics, Forschunginstitute Borstel, Borstel, Germany

Escherichia colilow molecular mass penicillin-binding

pro-teins (PBPs) include PBP4, PBP5, PBP6 and PBP6b

Evi-dence suggests that these proteins interact with the inner

membrane via C-terminal amphiphilic a-helices

Nonethe-less, 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 a-helices

Con-sistent withthese predictions, Fourier transform infrared

spectroscopy (FTIR) has shown that peptide homologs of

the PBP4 and PBP5 C-terminal regions, P4 and P5,

respectively, bothpossessed the ability to adopt a-helical

structure in the presence of lipid However, whereas P4 appeared to show a preference for interaction with the sur-face regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine mem-branes Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal a-helices, the PBP5 and PBP6 C-terminal a-helices may possess hydrophobicity gradients and penet-rate membranes in an oblique orientation

Keywords: penicillin-binding protein; C-terminal a-helix; hydrophobicity gradient; membrane

The Escherichia coli, low molecular mass penicillin-binding

proteins (PBPs) include PBP4, PBP5, PBP6 [1,2], PBP6b [3],

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

sugges-ted for PBP6b [3] Nonetheless, hydropathy plot analysis for

each of these membrane-associated PBPs shows no

con-ventional 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 [15] 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 a-helical structure [16] Furthermore, incorporation of a proline residue into the PBP5 C-terminal region, with its ability to disrupt or distort a-helical structure, greatly destabilized the membrane anchoring of the protein [17] whilst fusion of the PBP5 C-terminal region to a periplasmic b-lactamase led to

a membrane bound form of the enzyme [18]

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 [21] have predicted that the PBP5 and PBP6 C-terminal regions would form strongly amphiphilic a-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 [22] and showed high levels of lipid monolayer penetration [23] In contrast, similar theoretical analyses have predicted that the PBP4 and PBP6b C-terminal regions would form weakly amphiphilic a-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 [22] and low levels

of lipid monolayer penetration [23] In addition, hydropho-bic moment profile analysis has predicted that the PBP4 C-terminal region possesses an almost equal potential to

Correspondence to 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: daphoenix@uclan.ac.uk

Abbreviations: Myr 2 -PGro, dimyristoylglycerophosphoglycerol;

Myr 2 -PCho, dimyristoylglycerophosphocholine; Myr 2 -PEtn,

dimyristoylglycerophosphoethanolamine; FTIR, Fourier transform

infrared spectroscopy; PBPs, penicillin-binding proteins; SUV, small

unilamellar vesicle.

Enzymes: Escherichia coli PBP4 (EC 3.4.16.4; EC 3.4.99-);

E coli PBP5 (EC 3.4.16.4); E coli PBP6 (EC 3.4.16.4);

E coli PBP6b (EC 3.4.16.4).

(Received 10 May 2002, revised 24 September 2002,

accepted 7 October 2002)

interact with membranes via either amphiphilic b-sheet or

amphiphilic a-helical secondary structures [19] 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

a-helices in their C-terminal regions Recently confirmed by

experimental results [25,26], these are a class of a-helices

whose lipid interactions are predicted to involve penetration

of the membrane at a shallow angle due to a hydrophobicity

gradient along the a-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

orienta-ted a-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

transi-tion temperature and membrane fluidity of membranes

formed from either: Myr2-PGro, Myr2-PCho or Myr2-PEtn

M A T E R I A L S A N D M E T H O D S

The identification of candidate oblique orientated

a-helix forming segments

The primary sequence of the influenza viral fusion peptide,

HA2, a known oblique orientated a-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 [34] The

hydropho-bicity of successive amino acids in these sequences are

treated as vectors and summed in two dimensions, assuming

an amino acid side ch ain periodicity of 100 The resultant

of this summation, the hydrophobic moment, lH, provides

a measure of a-helix amphiphilicity Our analysis used a

moving window of 11 residues and for eachsequence under

investigation (Table 1), the window with the highest hydrophobic moment was identified (Table 1) For these windows, the mean hydrophobic moment, ÆlHæ, and th e corresponding mean hydrophobicity,ÆH0æ (Table 1), were computed using the normalized consensus hydrophobicity scale of Eisenberg et al [35] and plotted on the hydrophobic moment plot diagram [36], as modified by Harris et al [28] (Fig 1)

Table 1 The primary sequences and hydrophobic moment parameters of protein segments The C-terminal sequences of PBP4 [44], PBP5 and PBP6 [45], PBP6b [3] and the primary sequence of the HA2 fusion peptide [33] were analyzed using hydrophobic moment methodology [34] Eleven residue windows of maximum amphiphilicity were identified (shown in bold) and are shown, along with their corresponding Æl H æ and ÆH 0 æ.

Fig 1 The hydrophobic moment plot diagram The conventional hydrophobic moment plot diagram of [36] with an overlaid gray region delineating candidate oblique orientated a-helices [28] are shown The sequences shown in Table 1 were plotted on the diagram according to their Æl H æ and corresponding ÆH 0 æ values (Table 1) Data points rep-resenting th e C-terminal sequences of PBP5 (2) and PBP6 (3) can be seen to lie in the gray region, proximal to that representing the HA2 peptide (5), indicating that these C-terminal sequences may be candi-dates for oblique orientated a-helix formation The data points representing the C-terminal sequences of PBP4 (1) and PBP6b (4) lie outside this area and are unlikely to adopt such structure.

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 1Maqueous stock

solutions at 4C 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

accord-ing to Keller et al [37] Essentially, lipid/chloroform

mixtures were dried withnitrogen 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 4C witha Soniprep 150 sonicator

(amplitude 10 lm) until clear suspensions resulted (30

cycles of 30 s), which were then centrifuged (15 min,

3000 g, 4C)

FTIR conformational analyses of P4 and P5

To give a final peptide concentration of 1 mM, eith er 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 CaF2crystal, and the free excess

water was evaporated at room temperature The single

band components of the P4 or P5 amide I vibrational

band (predominantly C¼O stretch) was monitored using

an FTIR Ô5-DXÕ 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 widthand 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

suchas Fourier self-deconvolution [38] 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

ResearchCouncil, Ottowa, Canada) An estimation of the

number of band components was obtained from

deconvo-lution of the spectra, curve fitting was then applied within

the original spectra after the subtraction of baselines

resulting from neighboring bands Similar to the

deconvo-lution 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 TheCURFITprocedure 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 a-helical structure (1650–1655 cm)1) and

b-sheet structures (1625–1640 cm)1) were computed and are

sh own in Table 2

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 inves-tigated 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-lm thick Teflon spacer and subjected to automatic temperature scans witha heating rate of 3C

5 min)1within the temperature range 0 to 60C For every

3C interval, 50 interferograms were accumulated, apo-dized, Fourier transformed and converted to absorbance/ temperature spectra [39] (Figs 3 and 6) These spectra monitored changes in the bfi a 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, ms(CH2), which is known

to be a sensitive marker of lipid order The peak position of

ms(CH2) lies at 2850 cm)1in the gel phase and shifts at a lipid specific temperature Tcto 2852.0 cm)1)2852.5 cm)1in the liquid crystalline state

R E S U L T S

The identification of candidate oblique orientated a-helix forming segments

The sequences shown in Table 1 were plotted on the modified hydrophobic moment plot diagram (Fig 1) according to their ÆlHæ and ÆH0æ 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

Table 2 P4 and P5 secondary structural levels in the presence of lipid Levels of secondary structure determined for P4 and P5 FTIR con-formational analysis of P4 and P5 were performed witheachpeptide either: in aqueous solution (–) or in the presence of either: dimyristoyl phosphatidylcholine (Myr 2 -PCho), dimyristoyl phosphatidylethanol-amine (Myr 2 -PEtn), or dimyristoyl phosphatidylglycerol (Myr 2 -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.

Peptide Lipid

a-helical structures (%)

b-sheet structures (%)

orientated a-helix former These observations indicate that the PBP5 and PBP6 C-terminal sequences are candidate oblique orientated a-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 a-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 suchas the ester double bond stretching at 1738 cm)1, th e meth ylene ch ain scissoring mode at 1464 cm)1 , and the phosphate antisymmetric stretching at 1240–1200 cm)1, and the peptide bands, amide

I (predominantly C¼O stretching) and amide II (predo-minantly 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 b-sheet structures, ranging from 63% in the presence of Myr2-PEtn to 85%

in the aqueous peptide Nonetheless, the peptide adopted significant levels of a-helical structure in the presence of bothMyr2-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 a-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 Tcof bothMyr2-PGro and Myr2 -PCho membranes as 25C and that of Myr2-PEtn mem-branes as 47C (Figs 3A–C and 6A)C) In the presence of P4, no significant changes in either the membrane fluidity or the Tcof 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

Fig 2 FTIR conformational analyses of P5 in the presence of lipid (A–D) show FTIR conformational analyses of P5 in the presence of Myr 2 -PCho, Myr 2 -PEtn and Myr 2 -PGro and in aqueous solution, respectively In eachcase, the major contribution to P5 came from a-helical structure (1650–1655 cm)1) although significant levels of b-sheet structures (1625–1640 cm)1) can also be seen In all cases, annotated numbers indicate band peak absorbances.

have a significant effect on the Tcof the lipid, with Tcbeing recorded as 42C in the presence of the peptide (Fig 3B) The presence of P5 had a strong effect on the Tc and membrane fluidity of bothMyr2-PCho and Myr2-PEtn membranes with Tc being recorded as 13 and 42C, 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

30C rather than the 25 C of the pure lipid (Figs 3C and 6C)

D I S C U S S I O N

Here, we analyzed the C-terminal sequences of PBP4, PBP5, PBP6 and PBP6b to identify candidates withthe potential

to form oblique orientated a-helices [27,30] and based on theirÆlHæ , and ÆH0æ 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 a-helices However, the C-terminal regions of PBP5 and PBP6 were predicted to be candidates for the formation of such a-helices and are similar to the viral fusion peptide, HA2 (Fig 1), a peptide shown to penetrate membranes via an oblique orientated a-helix The C-terminal a-helices of PBP5 and PBP6 show many structural resemblances to the HA2 oblique orientated a-helix It can be seen from Fig 7 that each of these a-helices possesses a wide hydrophobic face, which includes bulky tryptophan, phenylaniline and isoleucine amino acid residues, and a glycine rich hydrophilic face These struc-tural features give a-helices an effective wedge shape, which appears to be utilized by HA2, and a number of other oblique orientated a-helix forming peptides, to destabilize membranes, leading to membrane fusion [40,41] Further-more, Roberts et al [21] analyzed the PBP5 and PBP6 C-terminal a-helices according to DWIH methodology and

Fig 3 FTIR lipid phase transition analysis of P4 (A–C) show the

effect of P4 on the b fi a acyl chain melting behavior of Myr 2 -PCho,

Myr 2 -PEtn and Myr 2 -PGro membranes, respectively, which were

monitored by FTIR spectroscopy as a function of temperature The T c

of Myr 2 -PCho membranes alone (j) was recorded as 25 C and in th e

presence of P4 (d), no significant changes in either the T c or the

membrane fluidity of the membrane was detected (A) The T c of Myr 2

-PEtn membranes alone was recorded as 47 C (j) and although in the

presence of P4 (d), no significant effect on the fluidity of the membrane

was detected, the T c of the membrane was recorded as 42 C (B) Th e

T c of Myr 2 -PGro membranes alone (j) was recorded as 25 C wh ilst

in th e presence of P5 (d), phase transition occurred over the interval

20 C to 30 C accompanied by an increase in gel phase fluidity and a

decrease in liquid crystalline phase fluidity (C).

Fig 4 FTIR overview spectrum of P4 in the presence of Myr 2 -PCho The peak absorbances for lipid vibrational bands such as the ester double bond stretching at 1738 cm)1, th e meth ylene ch ain scissoring mode at 1464 cm)1, and the phosphate antisymmetric stretching at 1240–1200 cm)1, and the peptide bands amide I (predominantly C¼O stretching) and amide II (predominantly N–H bending) are shown.

showed that the nature and order of the amino acid residues forming these a-helices were highly significant This is consistent withthe ordered spatial arrangement of amino acid residues necessary to maintain the hydrophobicity gradients of oblique orientated a-helices [29] In contrast to the PBP5 and PBP6 C-terminal regions, it can be seen from Fig 7 that, in an a-helical conformation, the C-terminal regions of PBP4 and PBP6b show ill-defined hydrophobic faces and few structural resemblances to the HA2 a-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 conforma-tional analysis showed that both in aqueous solution and

in the presence of each lipid examined, over 60% of P4 architecture was formed from b-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 a-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 a-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) BothP4 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 withlipid membranes is related to the ability of each peptide to adopt amphiphilic a-helical structure Furthermore, these results are consistent with those of Brandenburg et al [42] and suggest that the ability of P4 to adopt suchstructure may be related to the character-istics of the interface rather than solely the lipid type Our FTIR lipid phase transition analyses showed that the presence of bothP4 and P5 led to a broadening of the Tcof Myr2-PGro membranes (25C) withphase transition occurring over a temperature range (20–30C) accompan-ied 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 [43] 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 fluidiza-tion of the gel phase and rigidificafluidiza-tion of the liquid

Fig 5 FTIR conformational analyses of P4 in the presence of lipid (A–D) show FTIR conformational analyses of P4 in the presence of Myr 2 -PCho, Myr 2 -PEtn, Myr 2 -PGro and in aqueous solution, respectively In eachcase, the major contribution to P4 came from b-sheet structures (1625–1640 cm)1) Significant levels of a-helical structure (1650–1655 cm)1) can be seen in the presence of Myr 2 -PEtn (B) and Myr 2 -PGro (C) but there is no evidence of such structure in the presence of Myr 2 -PCho (A) or in aqueous solution (D) In all cases, annotated numbers indicate band peak absorbances.

crystalline phase These results do not necessarily mean that P4 and P5 interact withthe Myr2-PGro acyl chains region and in isolation, do not allow a clear interpretation as to the nature of P4 and P5 interaction withMyr2-PGro mem-branes 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 5C 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 mem-branes (Fig 6B) withthe presence of the peptide leading to increased membrane fluidity in bothcases, accompanied by decreases in membrane Tc of 12 and 5C, respectively Taken overall, these results clearly show that there are fundamental differences between the mechanisms of mem-brane 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 a-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 penetra-tion could explain the high levels of hemolysis shown by both this peptide and P6, a peptide homolog of the PBP6 C-terminal region [22] for HA2 is hemolytic yet abolition of the peptide’s hydrophobicity gradient leads to loss of hemolytic and fusogenic ability [31] 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 suchan interaction would be weak and unlikely to play a

Fig 6 FTIR lipid phase transition analysis of P5 (A–C) show the

effect of P5 on the b fi a acyl chain melting behavior of Myr 2 -PCho,

Myr 2 -PEtn and Myr 2 -PGro membranes, respectively, which were

monitored by FTIR spectroscopy as a function of temperature The T c

of Myr 2 -PCho membranes alone (j) was recorded as 25 C wh ilst in

the presence of P5 (.) th e T c of the membrane was recorded as 13 C,

accompanied by an increase in membrane fluidity (A) The T c of Myr 2

-PEtn membranes alone (j) was recorded as 47 C whilst in the

presence of P5 (.) th e T c of the membrane was recorded as 42 C,

accompanied by an increase in membrane fluidity (B) The T c of Myr 2

-PGro membranes alone (j) was recorded as 25 C whilst in the

presence of P5 (.), phase transition occurred over the interval 20 to

30 C accompanied by an increase in gel phase fluidity and a decrease

in liquid crystalline phase fluidity (C).

Fig 7 Helical wheel representations of protein segments The C-ter-minal sequences of PBP4, PBP5, PBP6 and PBP6b, and the primary sequence of the HA2 fusion peptide (Table 1) modeled as a-helices according to the methodology of Schiffer and Edmundson [46], assu-ming an angular periodicity of 100 are shown The a-helices of HA2, PBP5 and PBP6 show well defined amphiphilicity and, in common, possess glycine rich hydrophilic faces with wide hydrophobic faces rich

in bulky amino acid residues The a-helices of PBP4 and PBP6b show ill-defined faces and few structural resemblances to that of HA2.

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 [12] and

protein–protein interactions [8–11]

In summary, our results show that the PBP4 C-terminal

sequence is able to adopt a-helical and b-sheet structure in

the presence of lipid and may weakly associate with the

membrane lipid headgroup region via predominantly

elec-trostatic interactions In contrast, our results suggest that

the PBP5 C-terminal region possesses a strong intrinsic

tendency to bothadopt a-helical structure and to penetrate

the membrane hydrophobic core region It appears that this

C-terminal a-helix, and that formed by PBP6, possess

hydrophobicity gradients, which we suggest may facilitate

membrane penetration in an oblique orientation

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