Application of biophysical techniques in field of petide interactions with lipid membranes antimicrobial peptides cd.

2. BIOPHYSICAL INVESTIGATION

2.1. CIRCULAR DICHROISM SPECTROSCOPY

To get an insight into the molecular structure of an investigated antimicrobial peptide CD spectroscopy technique was employed. Circular dichroism is the phenomenon observed when a plane polarized light transverses through a chiral sample which absorbs at the frequency of the incident radiation. The difference in absorption between right and left circularly polarized light forms the basis of the measurement. In a single CD experiment the sample is illuminated with beam of a plane polarised light (which can be considered as the superposition of left and right circularly polarised light). Plane polarised light, when passing through the chiral environment, experiences different absorption for left and right circularly polarised components of plane polarised beam resulting in elliptical polarization on the output. As the result the value of sample’s ellipticity can be plotted versus the wavelength of the illuminating beam giving the CD spectra. Differences in the protein’s secondary structure CD spectra in far UV region enable the determination of the folding patterns of whole protein.

From the investigated peptide’s amino-acid sequence analysis one could expect that in the solution an α-helical fragment might be formed. Such helix would have two surfaces – one hydrophobic and another one hydrophilic as shown on Fig.2 [6].

However CD spectra of the peptide in aqueous solution revealed an unordered character of the investigated AMP (Fig. 3). Upon the addition to the negatively charged DMPC/DMPG (2:1 molar ratio) liposome suspension major changes in AMP’s structure were observed.

Results obtained from the peptide’s CD spectra analysis (carried out with CDPro software developed by R.W. Woody) displayed a significant increase in peptide organization (major increase in α-helix content). Such behaviour suggests a kind of peptide “activation” in the lipid membrane proximity.

2.2. DIFERENTIAL SCANNING CALORIMETRY

In order to monitor the changes in model lipid membrane upon antimicrobial peptide addition calorimetric measurements were carried out. The structure of lipid membrane is maintained by the cooperative interactions of many weak or inter- and intramolecular forces. These highly cooperative structures undergo conformational transitions on being heated/cooled, and significant information concerning the forces involved can be derived from differential scanning calorimetry (DSC) studies of these transitions. In DSC experiment the differential power (heat) required to maintain the given heating (cooling) rate of a sample (in a suitable solvent) at the same value as the solvent in the reference cell is measured. Upon heating of the liposome containing sample two phase transitions peaks are visible, corresponding to the anionic DMPG and zwitterionic DMPC respectively (Fig 4). After antimicrobial peptide addition to the liposome containing sample visible changes in phase transition profile occur. The transition enthalpy decreases and we cannot distinguish the DMPG from DMPC transition anymore. These changes reflect the disorganisation in the hydrophobic part of the lipids and significant lack of cooperation between them. Clearly the lipid membrane properties are disrupted.



2.3. HIGH RESOLUTION NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Use of the combined approach of wideline and high resolution magic angle spinning (MAS) NMR spectroscopy on membrane systems enables observations of the peptide interactions with liposomes on a molecular level [5]. The natural abundance of 14N and 31P in phospholipids building the model membrane has been used for wideline NMR investigation of phase behaviour of lipid system upon addition of AMP. To increase the resolution of wideline NMR spinning of the sample at the magic angle has been used to average the chemical shift anisotropy of lipid phosphates. The 31P MAS NMR spectra of lipid membrane and the membrane incubated with antimicrobial peptide for 20 days are shown on Fig. 5 (left and right panel respectively).


In the AMP containing sample, instead of two peaks corresponding to phosphate resonances from DMPC and DMPG one isotropic peak appears reporting the fast movement of lipid headgroups. Changes in resonance intensities are more intense for the anionic lipid, indicating that the electrostatic interactions between negative lipid headgroups and the cationic peptide residues are involved.