Peptides targeted by intrinsic lipidation in vitro do not undergo post-translational lipidation, therefore knowledge of their structure and properties following acylation is limited. Lytic peptide melittin is well characterised in solution and membrane bound forms, however its intrinsically lipidated analogues have received little scientific focus.198 Properties of acylated melittin, in comparison to its unmodified counterpart, were therefore probed in order to better understand their biological relevance. The advent of N -stearylated melittin used as a gene transfection vector, serves to highlight the need for improved understanding.199 Four acylated
82
Chapter 4. Peptide Intrinsic Lipidation 83
melittin analogues were selected for study, N palmitoyl melittin, K23palmitoyl melittin, N -oleoyl melittin and K23--oleoyl melittin. Species mirror the most common acylation sites and fatty acid chains identified during study of intrinsic lipidation within eukaryotic membranes.
4.2.1 Structure of Acylated Melittin
Solution phase melittin adopts a random coil configuration of undefined structure at low concentration, and a tetrameter shielding hydrophobic residues from the aqueous external envi-ronment at high concentration.200–202 Increasingly hydrophobic acylated melittin is predicted to exhibit modified structural behaviour in aqueous solution, shielding the fatty acid chain from the unfavourable external environment. Fatty acid chain chemistry and modification position may influence this solution phase structural behaviour. Biophysical techniques were therefore employed in order to probe the solution phase structure of four acylated melittin derivatives, N -palmitoyl melittin, K23-palmitoyl melittin, N -oleoyl melittin and K23-oleoyl melittin.
250 300
-300 -200 -100 0 100 200
Wavelength (nm)
Molar Ellipticity (degcm2/dmol)
Figure 4.1 CD spectra of solution state melittin (black); N -palmitoyl melittin (red); K23-palmitoyl melittin (orange); N -oleoyl melittin (blue); K23-oleoyl melittin (green).
Circular dichroism (CD) creates a characteristic spectrum based upon peptide interactions with left circularly polarised light and right circularly polarised light. Comparison to literature standards then facilitates identification of peptide secondary structure.203,204 CD spectra of four acylated melittin derivatives in aqueous solution, Fig. 4.1, exhibit considerable variation from the random coil of solution phase melittin. Increased negative dichroism at approximately 220 nm suggests acylated melittin derivatives adopt an α-helical conformation in solution.
Within error, % helicity is consistent across all forms of acylated melittin, suggesting secondary structure formation occurs irrespective of acyl chain chemistry or position. Comparison to α-helical structures of unmodified melittin suggests the solution phase conformation of acylated
84 4.2. Characterisation of Acylated Melittin
melittin is α-helical with a central kink attributed to the proline residue. However, it is unclear whether bulk peptide structure mimics the individual helices of membrane bound unmodified melittin, or aggregates, mirroring the tetrameric structure observed in solution at high melittin concentrations.205,206
The solution phase structure of acylated melittin derivatives can be further probed by employing the intrinsic fluorescence of aromatic amino acid residue tryptophan. Tryptophan emission is susceptible towards external environment, undergoing a blueshift in response to increased local hydrophobicity. Spontaneous melittin helix formation is characterised by a blueshift in maximum tryptophan fluorescence from 352 nm to 336 nm.45,62 The extent of helix formation can therefore be approximated by the I(336 nm)/I(352 nm) ratio. Analogous tryptophan fluorescence behaviour is predicted for acylated melittin, such that helicity can be determined by the I(336 nm)/I(352 nm) ratio. Fig. 4.2 shows tryptophan emission following excitation at 280 nm for melittin and N -acylated analogues. K23-acylated melittin species have been omitted for clarity, however spectra resemble those of their N -acylated counterparts. Acylated melittin species exhibit a blueshift in emission maxima compared to unmodified melittin, suggesting tryptophan resides in an increasingly hydrophobic environment. The I(336 nm)/I(352 nm) ratio is presented for solution phase melittin species in Table 4.1. Increased ratios are observed for all acylated species compared to melittin, supporting tryptophan residence in the increasingly hydrophobic environment associated with α-helix formation. The I(336 nm)/I(352 nm) ratio is not observed to be dependent upon either acyl chain chemistry or modification position.
Furthermore, the origin of the heightened I(336 nm)/I(352 nm) ratio attributed to N -oleoyl melittin remains unclear.
300 350 400 450
0 20 40 60 80 100
Wavelength (nm)
Intensity(%)
Figure 4.2 Tryptophan emission following excitation at 280 nm for melittin (solid black line); N -palmitoyl melittin (dotted and dashed red line); N -oleoyl melittin (dashed blue line).
Chapter 4. Peptide Intrinsic Lipidation 85
Peptide Emission Maximum (nm) I(336 nm)/I(352 nm)
Melittin 362.5 0.67
N-palmitoyl melittin 355.5 0.74
K23-palmitoyl melittin 355.5 0.77
N-oleoyl melittin 354.5 0.86
K23-oleoyl melittin 356.5 0.73
Table 4.1 Emission maxima and I(336 nm)/I(352 nm) ratio for solution phase melittin analogues based upon tryptophan emission following excitation at 280 nm.
Combined CD and trytophan emission data suggest solution phase acylated melittin adopts an α-helical structure. Based upon the constrained nature of secondary amino acid proline, combined with similarities between CD of solution phase acylated melittin and membrane bound unmodified melittin, it is further speculated that the structure contains a kink at proline. Comparison of palmitoyled and oleoylated peptides suggests adopted structure is not influenced by modification position or acyl chain length. However, literature studies suggest that more significant reductions in carbon chain length may favour random coil formation in favour of an α-helix.207 Similarly, internal acyl chain modifications may exhibit structural differences compared to the N -terminal and K23 modified species tested.
4.2.2 Critical Micelle Concentrations for Acylated Melittin
Acylated melittin, comprised of a hydrophobic fatty acid chain and hydrophilic cationic C-terminus, can be characterised as amphiphilic. Amphiphilic molecules often aggregate in aqueous solution, in order to promote the maximum number of favourable hydrophobic and hydrophilic interactions. Micelles, depicted in Fig. 4.3, are spherical amphiphilic aggregate structures adopted by detergents, including lysolipids.208 Micelle formation requires substrate concentrations above a critical micelle concentration (CMC), below which only monomers of undefined structure exist. Naturally lipidated peptides have been reported to undergo micellisation in aqueous solution at concentrations above 0.1 µm.209,210Biologically relevant examples are summarised in Table 4.2. Therefore, whilst unmodified melittin does not exhibit micelle formation in aqueous solution, it may be induced by the increased hydrophobicity of acylated melittin analogues.
86 4.2. Characterisation of Acylated Melittin
Figure 4.3 Schematic representation of a typical micelle.
Lipidated Peptide CMC Arthrofactin 10 µm Surfactin 70 µm Pseudofactin II 130 µm
Table 4.2 CMC of known lipidated peptides available from literature sources.209,210
Acylated melittin micellisation was studied over the concentration range 100 µm to 1 µm using a Rhodamine 6G assay. Selected due to the cationic nature of melittin, Rhodamine 6G emission at 550 nm is quenched upon micelle formation due to the localised hydrophobic environment.211,212 Four acylated melittin analogues were tested, N -palmitoyl melittin, K23-palmitoyl melittin, N -oleoyl melittin, and K23-oleoyl melittin, in order to distinguish CMC variation dependent upon substitution location or acyl chain chemistry. Rhodamine 6G emission in the presence of palmitoylated melittin derivatives, Fig. 4.4, exhibits quenching associated with micelle formation. In the presence of N -palmitoylated melittin, Rhodamine 6G emission decreases upon initial peptide addition, indicating immediate micelle formation, and a CMC value of 1 µm or less. In contrast, the observed emission profile in the presence of K23-palmitoyl melittin is reminiscent of literature studies, with an initial plateau followed by a decrease denoting the CMC at 5 µm.211 Calculated CMC values for palmitoylated melittin derivatives are low compared to the lipidated peptides evidenced in Table 4.2. Observed values fall in a similar concentration range to CMC of lysolipid, for example the CMC of PPC reported in the range 4 µM to 8.3 µM.213
Rhodamine 6G emission in the presence of oleoylated melittin derivatives, Fig. 4.5, exhibited different behaviour compared to their palmitoylated counterparts. N -oleoyl melittin is not observed to quench Rhodamine 6G emission over the concentration range studied, suggesting lack of micelle formation. Emission of Rhodamine 6G in the presence of K23-oleoyl melittin proves more challenging to interpret. A slight decrease in Rhodamine 6G emission is observed, however it is unclear as to whether this can be attributed to micelle formation, or to background emission variation. Attributing a CMC to K23-oleoyl melittin gives the value 7.5 µm, within the predicted concentration range for palmitoylated melittin and naturally acylated peptides.209,210
Chapter 4. Peptide Intrinsic Lipidation 87
Furthermore, a K23-oleoyl melittin CMC of 7.5 µm fits with observed lysolipid CMC trends, in which unsaturated species exhibit higher CMC than their saturated counterparts.33,214 However, attributing the observed decrease in Rhodamine 6G emission in the presence of K23-oleoyl melittin to background variation suggests oleoylated melittin derivatives do not undergo micelle formation. This observed distinction between palmitoylated and oleoylated species can be attributed to the reduced packing ability of unsaturated acyl chains.
0 50 100
0 50 100
Concentration (µM)
Intensity(arb.units)
Figure 4.4 Rhodamine 6G emission at 550 nm in the presence of increasing concentrations of N -palmitoyl melittin (solid red); K23-palmitoyl melittin (dashed orange). Vertical line denotes the CMC of K23-palmitoyl melittin at approximately 5 µm.
0 50 100
0 50 100
Concentration (µM)
Intensity(arb.units)
Figure 4.5 Rhodamine 6G emission at 550 nm in the presence of increasing concentrations of N -oleoyl melittin (solid blue); K23-oleoyl melittin (dashed green). The vertical line indicates the possible CMC of K23-oleoyl melittin at approximately 7.5 µm.
4.2.3 Antimicrobial Properties of Acylated Melittin
Naturally lipidated peptides exhibit bacterial toxicity associated with membrane disruption caused by their fatty acid substitutions. Toxicity can be quantified by Minimum Inhibitory Concentration (MIC), defined as the minimum concentration required to inhibit microorganism growth. MIC values attributed to lipidated peptides fall within the concentration range of
88 4.2. Characterisation of Acylated Melittin
0.5 µm to 20 µm.72 Unmodified melittin boasts MIC of 5.6 µm for S. aureus and 11.3 µm for E. coli, therefore acylated melittin is predicted to exhibit significant bacterial toxicity.215 This theory was probed by testing the antibacterial activity of four acylated melittin derivatives, N-palmitoyl melittin, K23-palmitoyl melittin, N -oleoyl melittin, and K23-oleoyl melittin.
The MIC for acylated melittin derivatives were determined against gram-negative E. coli following 16 hour incubation with serial peptide dilutions of 32 µm to 0.5 µm.216 Optical density measurements were used to determine the extent of bacterial growth compared to an untreated control, with complete growth inhibition defining the MIC. Unrestricted bacterial growth was observed in the presence of all acylated melittin analogues at concentrations below 32 µm, suggesting a lack of bacterial toxicity. At 32 µm inhibition was observed for all species, as shown in Table 4.3, with the exception of K23-oleoyl melittin. However, insufficient bacterial growth inhibition was observed to consider this concentration the MIC. Acylated melittin species are therefore noted to exhibit decreased E. coli toxicity compared to unmodified melittin, which has an MIC in E. coli of 11.3 µm.215 Bacterial growth relative an untreated control is determined to be 74 % and 77 % for N -palmitoyl melittin and K23-palmitoyl melittin respectively, indicating a slight inhibition increase attributed to the N -terminal modification.
N-oleoyl melittin exhibits 84 % E. coli growth compared an untreated control, suggesting oleoyl modifications are associated with reduced antimicrobial activity compared to their palmitoylated counterparts, despite increased hydrophobicity.
Peptide E. coli S. aureus N-palmitoyl melittin 74 % 90 % K23-palmitoyl melittin 77 % 100 %
N-oleoyl melittin 84 % 100 % K23-oleoyl melittin 100 % 100 %
Table 4.3 % bacterial growth observed for E. coli and S. aureus treated with 32 µm acylated melittin analogues. 100 % growth was determine by normalisation to untreated controls.
Analogous studies were conducted in order to determine acylated melittin induced growth inhibition over the concentration range 32 µm to 0.5 µm for gram-positive S. aureus. Measured optical densities suggested a lack of bacterial growth inhibition for all acylated peptide derivatives across the concentration range tested, as shown in Table 4.3, with the exception of 32 µm N -palmitoyl melittin. N -palmitoyl melittin at 32 µm exhibits 90 % S. aureus growth compared to an untreated control, highlighting the increased toxicity of this acylated derivative compared to its counterparts. Antimicrobial activity of lipidated melittin in gram-positive bacteria is therefore decreased compared to unmodified melittin, which has a MIC of
Chapter 4. Peptide Intrinsic Lipidation 89
5.6 µm.215 Observed toxicity correlates with E. coli data, suggesting N -terminal modification and palmitoylation promote antimicrobial activity compared to alternative locations and chain chemistries.