3.1. Antimicrobial Activity
The antimicrobial activities of LfB6 and C6-LfB6 were tested against a Gram negative (E. coli
ATCC 25922) and a Gram positive (S. aureus
ATCC 29213) bacterial strain using a standard microdilution Mueller Hinton broth assay adapted for cationic AMPs by Hancock, et al.
] This assay provides a stringent test, and therefore more clinically relevant results, for the activity of cationic antimicrobial peptides due to the high ionic strength of Mueller Hinton medium that can inhibit the activity of such peptides.[45
] The results, shown in , reveal that addition of the C6 acyl chain to the N-terminal of LfB6 did not increase the activity against Gram negative E. coli
; whereas the activity against the Gram positive strain S. aureus
was increased 3-fold compared to LfB6. Additionally, although both LfB6 and C6-LfB6 inhibited the growth of E. coli
(MIC 150 μ
g/ml), neither peptide was bactericidal. By contrast, both LfB and C6-LfB were bactericidal against S. aureus
at their respective MIC values of 150 and 50 μ
g/ml. Acylation of LF12, a 12 amino acid fragment from human lactoferricin, was found to increase the activity against both Gram positive and negative bacteria, however the improvement was higher for S. aureus
than for E. coli
]. The same study also found that the antimicrobial activity of the acylated peptides depended on the composition of the bacterial culture medium, with higher MIC values (lower activity) observed for assays performed with complex LB medium compared to those with low ionic strength buffer. Direct comparisons of the antimicrobial activities reported from various studies, therefore, are difficult since different bacterial strains and experimental protocols are frequently used.[46
] The lack of an outer membrane in Gram positive bacteria might explain the enhanced activity of the acylated peptide, C6-LfB6, against S. aureus
. The outer cell membrane of Gram negative bacteria poses an additional barrier for AMPs to gain access to the periplasmic space and inner cytoplasmic membrane. A systematic investigation into the effects of acylation on dermaseptin, a 13 amino acid AMP isolated from the South American tree frog of the Phyllomedusa
genus, revealed complex antimicrobial behavior.[47
] Enhanced activity against Gram positive S. aureus
was observed for intermediate chain length acyl (C6-C12) derivatives, whereas all acyl derivatives were detrimental to activity against E. coli
. It has been proposed that some cationic AMPs are retained on the outer membrane of Gram negative bacteria upon binding to lipopolysaccharide (LPS), thus impeding or precluding their access to the inner membrane.[48
] LPS-binding motifs consisting of two positively charged amino acid residues separated from the third by a short hydrophobic, aromatic stretch[50
] have been identified in human and bovine lactoferricin peptides.[51
] Limited access to the cytoplasmic membrane due to the absence of an outer membrane might also explain why both LfB6 and C6-LfB6 are bactericidal at their MIC values against S. aureus
, but not against E. coli
Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of LfB6 and C6-LfB6 (μg/ml)
3.2. Solid-State 2H NMR Spectroscropy
H-NMR is a well-established method for characterizing the liquid crystalline (Lα
) phase of lipid bilayers containing perdeuterated lipids because the segmental order parameters (SCD
) along the acyl chain C-D bonds can be estimated from the experimentally determined quadrupolar splittings (Δνq
). For samples aligned at the β
= 0 orientation the quadrupolar splitting and segmental order parameter are related by[52
is the quadrupolar coupling constant (QCC), which is ≈168 kHz for aliphatic C–D bond[54
]. The lipid acyl chain order of mechanically oriented lipid bilayers composed of sn-1
chain perdeuterated zwitterionic (POPC) or mixed anionic:neutral (POPE:POPG) lipids was examined by solid-state 2
H NMR in the presence or absence of C6-LfB6. The 2
H spectra, shown in , are characteristic of phospholipids in liquid crystalline bilayers, consisting of a series of doublet resonances resulting from the different CD2
segments along the lipid acyl chain[52
]. Rapid rotation of the terminal methyl group, in combination with its tetrahedral geometry results in low order parameters. The smallest quadru-polar splittings were therefore assigned to the terminal methyl group.[56
]. The remaining 2
H resonances were assigned to carbons in decreasing order along the phospholipid chain. The quadrupolar splittings vary from a minimum of about 5 kHz for the terminal methyl groups to a maximum of about 50 and 54 kHz for deuterons near the head group carboxyls of POPC and POPE:POPG, respectively. In the presence of 1 mol % C6-LfB6, the 2
H spectral width observed from POPC-d31
bilayers was slightly reduced compared to POPC-d31
bilayers without peptide (). Indeed the 2
H quadrupolar splitting was reduced at each position along the acyl chain, except for the terminal methyl group which was essentially unchanged; namely, the methyl group Δνq
was 4.7 kHz in the absence of peptide compared to 4.6 kHz in the presence of C6-LfB6. The maximum quadrupolar splitting in the plateau region resulting from the C2 position closest to the carbonyl group was reduced by approximately 2 kHz in the presence of C6-LfB6. Surprisingly, no changes were observed in the quadrupolar splittings for either POPE-d31
in the presence of C6-LfB6, as shown in , respectively. For the mixed POPE:POPG bilayers, the spectra are virtually superimposable in the presence and absence of peptide, regardless of which lipid is deuterated.
Figure 1 2H spectra of mechanically aligned bilayers composed of a) POPC-d31, b) POPE:POPG-d31 (3:1), and c) POPE-d31:POPG (3:1) at 50° C and β = 0. Solid lines are of pure lipid, dashed lines are in the presence of 1 mol % C6-LfB6. The 2H order (more ...)
The segmental order parameters along the lipid acyl chain, estimated from the quadrupolar splittings and plotted relative to the peptide-free control samples, are shown in . As noted above, there is little change in the order parameter profile for either the zwitterionic POPE or the anionic POPG when C6-LfB6 (1 mol %) is added to the mixed POPE:POPG (3:1) membranes The reduction in order induced in the zwitterionic POPC acyl chains by C6-LfB6 is largest near the lipid head group, extends into the plateau region toward the center of the bilayer, and tapers off near the terminal methyl group.
3.3. Solid-State 31P NMR Spectroscopy
To test whether C6-LfB6 influences the lipid phosphate head groups, the 31
P NMR chemical shift anisotropy (CSA) of multilamellar vesicles (MLVs) composed of POPC and POPE:POPG (3:1) was monitored in the presence and absence of peptide. The static 31
P spectra of POPC and POPE:POPG (3:1) bilayers, shown in , respectively, are characteristic of MLVs in the liquid crystalline phase (Lα
]. The CSAs of POPC (≈ 47 ppm ±
1 ppm) and POPE:POPG (3:1) (≈ 37 ppm ±
1 ppm) are furthermore unchanged in the presence of 1 mol % C6-LfB, indicating little observable perturbation of either the neutral or anionic lipid head groups. These findings support our previous results that acylated and Trp-methylated LfB peptides have little effect on the phosphate head groups of DMPC and DMPC:DMPG (3:1) bilayers even at 4 mol %[12
]. Moreover, the MD simulations also show no change.
Static 31P spectra of MLVs composed of a) POPC, b) POPE:POPG (3:1) at 50° C. Solid lines are of pure lipid, dashed lines are in the presence of 1 mol % C6-LfB6.
3.4. Membrane Order
The acyl C–H bond orientation relative to the membrane normal is expressed by the order parameter:
These order parameters can be measured experimentally by deuterium quadru-polar splitting in solid state NMR. The NMR experiment, however, cannot specify which order parameter is associated with which carbon, so the order parameters are typically sorted in decreasing order and it is assumed that this corresponds to increasing carbon number. When calculated from an MD simulation, it is known exactly which carbon is associated with which order parameter. In order to compare with the experimental results however, it is necessary to sort the simulation order parameters by decreasing magnitude.
The results of the tension titration on order parameters for the POPE:POPG neat system are shown in . Although the difference between 35.0 dyn/cm and 37.5 dyn/cm is minor, there is a greater difference with the 32.5 dyn/cm simulations. The 32.5 dyn/cm tension order parameters match the experimentally determined order parameters reasonably well. This tension was then used in all subsequent simulations. It is important to note that convergence of the area per lipid under a new tension (used as a criterion for determining the end of tension equilibration) took on average 100 ns; the standard deviation of the average areas for 200 ns trajectories was as large as 0.5 Å 2.
Figure 3 Order parameters for the simulation of neat POPE:POPG membranes at different tensions are shown here compared with the experimentally determined order parameters. The simulation order parameters are sorted to correspond to the experimental data. The first (more ...)
The order parameters for both neat and C6-LfB6 containing simulations are shown in . Row A shows the order parameters calculated from the simulation in their natural order while Row B shows them sorted for comparison against the experimental data, shown in Row C. In the POPE:POPG experiments, the POPE and POPG were deuterated separately and the individual order parameters are shown in the respective columns although the two lipids produce nearly identical values.
Figure 4 Order parameters for the different systems. Row A shows the order parameters in their “natural” order from the simulation. In Row B, they are sorted in decreasing order to match the NMR results. Row C shows the experimentally determined (more ...)
Comparing the order parameters from the simulation of POPE:POPG and the experimental data show that the profiles are very similar. Moreover, the experimental data shows virtually no change in order parameters upon C6-LfB6 binding—a result that is replicated in the simulations. This is perhaps a result of the low concentrations of acyl-peptides used.
Interestingly, the order parameters for the putative mammalian membrane (POPC) decrease noticeably upon association of C6-LfB6 in both the experiment and the simulation. This result seems counter-intuitive since AMPs typically affect mammalian membranes less than bacterial. This is surprising because one would expect the anionic “bacterial” POPE:POPG membrane to be more sensitive to peptide effects than the “mammalian” POPC membrane. Comparing the area per lipid between the POPE:POPG and POPC simulation (), the POPC membranes have a nearly 5 Å 2 greater area per lipid than the POPE:POPG membranes, perhaps as a result of the larger lipid head groups or the absence of inter-headgroup hydrogen bonds. The larger spacing between lipids could provide more opportunities for the C6-LfB6 to insert deep enough into the membrane to alter chain structure. The concentration of peptide used here (1:100) is far lower than what is needed to lyse membranes.
3.5. Acyl-Peptide–Membrane Association
Examining the contacts made between the different components of the acyl-peptide, such as the C6 tail, the arginines, and the tryptophans, and the system provides a quantitative measure of how the acyl-peptide associates with the membrane and where it is positioned once it is associated. shows the contacts made between C6-LfB6 and the POPE:POPG system. In panels A–C, the fractional contacts between each C6-LfB6 component and all atoms of POPE, of POPG, and all waters are shown, averaged over all C6-LfB6 molecules, i.e. 2 peptides × 4 repeats for POPC and 2×8 for POPE:POPG. The replicates for each simulation are needed to allow us to confidently discuss the mechanisms of binding, as any single trajectory may not have all of these features. In addition, the total lipid contact (sum of POPE and POPG contacts) is also shown. We started from an out-of-equilibrium state–no peptide bound–and noted the approach mechanisms. We define membrane association as the point where the lipid and water contacts are equal. Panel A shows the association of the C6 tails with the membrane occurring after approximately 75 ns. Panel B shows that the arginines associated very rapidly, within the first 25–50 ns. This is followed by the tryptophans, at approximately 50 ns. What is striking in these panels is that while the system consisted of a 3:1 ratio of POPE:POPG, the contacts made between C6-LfB6 and POPE are closer to twice that of POPG and are often almost equal, suggesting a preferential interaction between the peptide and PG. There is also only one instance of contact between two C6-LfB6 molecules, and it lasts for less than half of one trajectory, indicating that dimer formation is unlikely, at least at these concentrations. Overall, the C6 tails show the highest degree of contact with lipids (≈ 0.7), followed by the tryptophans (≈ 0.65) and the arginines (≈ 0.55).
Figure 5 Normalized contacts made between different parts of the C6-LfB and either water or lipid. The C6 tail is shown in Panels A and D, the Arginines in Panels B and E, and the Tryptophans in Panels C and F. The total lipid in Panels A, B, and C is the sum (more ...)
Panels D–F increase the detail of the contacts by breaking the lipids into their head groups (PE and PG) and the tails as separate entities, along with solvent. Panel D shows that the C6 tail inserts into the membrane and makes considerably more contacts with the acyl chains than the lipid head groups, indicating it is buried in the membrane. In contrast, the arginines, shown in Panel E, prefer polar contacts, remaining well solvated, and also making contacts with the PE and PG head groups as opposed to the acyl chains. This indicates that the arginines stay near the membrane interface. The tryptophans have nearly equal contacts with all components of the membrane and a lower solvent contact than the arginines. This suggests that the tryptophans can bury themselves slightly into the membrane, though not to the extent that the C6 tail inserts, and stay more near the membrane–water interface. This is consistent with much previous work suggesting tryptophans prefer to reside in the interface[59
The contacts for C6-LfB6 in POPC are shown in . Here, it is the C6 tail (Panel A) that rapidly associates with the membrane after approximately 25 ns, followed by the arginines (Panel B) and tryptophans (Panel C) at 50 ns. The overall pattern of contacts between parts of the C6-LfB6 and different components of the membrane are similar between the POPC and POPE:POPG systems. In both cases, the C6 inserts deeply into the membrane while the arginines remain near the membrane-solvent interface. The tryptophans also insert into the membrane, but not as deeply as the C6 tails. While it appears in that the tryptophans make more contacts with the PC head groups than the PE and PG headgroups in , the contacts in the latter are divided between two different headgroup species; comparing total head-group contacts, irrespective of whether it is with PE or PG, the pattern is similar between the POPC and POPE:POPG systems.
Contacts between different parts of the C6-LfB and either water or lipid. The C6 tail is shown in Panel A, the Arginines in Panel B, and the Tryptophans in Panel C.
Althought the contact plots suggest that the C6-LfB6 resides near the membrane-solvent interface and that the C6 tail reaches down into the membrane, it is not clear how far down the tail resides. A plot of the average centroid for the PE, PG, and PC head groups and the centroid of the C6 tails is shown in . The C6 tail inserts into the POPC membrane far faster than in the POPE:POPG mixture. Moreover, while the average location of the center of the lipid head groups is unchanged between POPC and POPE:POPG, the C6 tail is 1–2 Å deeper in the membrane in the POPC simulations. The greater depth of the C6 in POPC is consistent with there being more space between the lipids for the C6, and indeed the whole C6-LfB6.
Figure 7 Average distance from the membrane center (Z-coordinate) for the lipid head groups and the C6 “tail”. The tail is buried inside the membrane once C6-LfB binds. The wide bands show the average distance from the membrane center for C1 and (more ...)
There is a significant difference in the orientation of the C6 tails in the membrane for POPC compared with POPE:POPG. The orientation can be quantified by computing the principal axes for the heavy atoms of the C6. The first principal axis points along the direction of the tail, so we computed the cosine of the angle between it and the membrane normal. The probability distribution for the tail orientations in both POPC and POPE:POPG are shown in . In POPE:POPG, the C6 tail distribution is fairly flat until at about 0.6 (corresponding to 53°) leading to a peak at nearly 1 (or parallel to the membrane normal). In contrast, the POPC distribution is largely flat with a peak in the vertical orientation some 30% smaller than POPE:POPG. This is again consistent with there being greater area per lipid in POPC, providing more space for the C6 to adopt a larger range of orientations.
Probability distribution of the cosine of the angle between the C6 tail and the membrane normal. The error bars shown are the standard errors of the angles of individual peptides.
The distribution of the electron density for both the peptide backbone and the C6 tails is shown in . The density for the lipid head groups are shown as well: PE and PG in and PC in . In the POPC system, the peptide backbone resides approximately 2 Å deeper than in the POPE:POPG system. The C6 tail also resides approximately 2 Å deeper in the POPC system. In both systems, the tail can also reside at the membrane-water interface. Comparing the area under the peaks, the probability of finding the tail buried is three times as likely as at the interface in both POPC and POPE:POPG.
Probability distribution for the location of the lipid head groups, C6-LfB6 backbone, and C6 tail. Panel A shows the results for the POPE:POPG simulations while panel B shows the results for the POPC simulations.
Representative conformations of one of the bound peptides are shown in for the POPE:POPG system (Panel A) and POPC (Panel B), taken from approximately 300 ns into the respective simulations. In both cases, the C6 tail is inserted into the membrane with the arginines and tryptophans remaining near the head groups/membrane-water interface.
Figure 10 Representative conformations of bound C6-LfB6 in POPE:POPG (Panel A) and in POPC (Panel B). Membrane lipids are shown as gray surfaces with the head groups colored red (PE), green (PG), and yellow (PC). The peptide residues are colored slate (Arg), magenta (more ...)
3.6. Hydrogen Bonding
We looked at the fraction of time spent with a hydrogen bond between various possible donor nitrogens moeities in the acyl-peptide with the lipid carbonyls, lipid phosphates, solvent, and with the acyl-peptide itself. We found no significant change in hydrogen bonding patterns between the POPE:POPG system and the POPC system. On average, there is a hydrogen bond between the lipid phosphates and acyl-peptides one third of the time and with solvent another third of the time. The greatest contact between the acyl-peptides and the lipid phosphates occurred between the arginines (30%–40% of the time), followed by the tryptophans (18%–20%), and the glutamine (≈ 18%).
There are no significant changes in lifetimes found with the exception of the hydrogen bond between the indole-nitrogen of tryptophan 3 (closest to the C6) and the lipid carbonyls, shown in . In this case, there is a slightly longer lifetime for W3 in POPE:POPG than W5. The lifetime for the latter is also comparable to the lifetimes of both W3 and W5 in POPC.
Figure 11 Time correlation function for hydrogen bonding between the two different indole-nitrogens of the acyl-peptide and the lipid carbonyl oxygens is shown for both the POPE:POPG and POPC systems. The error bars represent the standard errors of the averages (more ...)