2.14 .1 Mas s sp ectro metry
Samples from the HPLC fractions were prepared by adding 10μL 10% Formic acid to 90 μL of the HPLC fraction. Samples were analysed using electrospray ionisation mass spectrometry (ESI-MS) on a Bruker MicrOTOF. The positive ionisation mode was used and detection was between 50 and 3000 m/z. Spectra were typically recorded for 2 minutes, averaged and deconvoluted to determine the mass of the main species.
2.14 .2 Cross -linking
Cross-linking of the peptides in different concentrations of detergent was performed with bis(sulfosuccinimidyl)suberate (BS3) (Pierce) or glutaraldehyde (TAAB) according to the manufacturer’s instructions. Typically a 40 μL reaction containing 40 µM peptide, a 50-fold excess of cross-linker and varying amounts of detergent in 20 mM sodium phosphate buffer pH 8.0, was incubated at room temperature for 30 min. Reactions were quenched with 2 µM Tris-HCl pH 7.5.
2.14 .3 Circul ar dich ro is m (CD)
Peptide samples were prepared with 50 mM sodium phosphate buffer pH 7.5, 100 mM NaCl and varying amounts of DPC (Avanti Polar Lipids) to a final concentration of 80 µM. Measurements were taken in a J-815 spectropolarimeter (Jasco UK, Great Dunmow, UK) using a 1 mm path-length quartz cuvette (Starna; Optiglass Ltd., Hainhault, UK). Circular dichroism (CD) spectra were recorded between 180 and 260 nm with a data pitch of 0.2 nm, a bandwidth of 2 nm, a scanning speed of 100 nm/min, and a response time of 1 s. Four scans were averaged and the spectrum of buffer without peptide was subtracted to give the final spectra. Spectra of peptide in liposomes were also measured. The high tension of the spectra was also recorded.
2.14 .4 Ori ented ci rcular di chrois m (OCD)
Lipid and peptide were codissolved in TFE with final concentrations of 10 mg/mL and 1 mg/mL respectively. The lipid and peptide solution was then dried under
nitrogen on a glass slide to form an even lipid-peptide multilamellar film. To ensure complete removal of the organic phase, the glass slide was left in a desiccator overnight. To hydrate the lipid bilayer the glass slide was placed in a custom built chamber overnight with a small amount of water. The slide was placed in a custom built orientable slide holder with the multilamellar film perpendicular to the incident light. Oriented CD (OCD) measurements were taken using the same parameters as for CD (§2.14.3) with a 10 nm bandwidth. To minimise artefacts arising from linear dichroism the slide was rotated 360 in the plane perpendicular to the incident light, taking measurements at 45º increments. The measurements were averaged and the spectrum of sample without peptide was subtracted to give the final OCD spectra.
2.14 .5 Linea r di ch rois m (LD)
Liposomes with 0.5 mg/mL peptide were prepared using 50 mM sodium phosphate buffer, pH 7.0. A 5:1 lipid to peptide ratio was used for the TatATMpeptide analysis.
Linear dichroism (LD) spectra were acquired between 180 and 360 nm at room temperature. An in-house built micro-volume Couette cell (Marrington et al., 2004; Marrington et al., 2005) was used; equivalent models are commercially available (Kromatek, Great Dunmow, UK). Samples were aligned by applying voltages of 3, 4 and 5 V, corresponding to 3k, 4k and 5k rpm. The acquisition parameters were the same as those for CD (§2.14.3). A spectrum of the non-aligned sample was subtracted from the aligned spectra to give the final LD signal. The spectra were corrected for light scattering due to the liposomes using the method given in (Nordh et al., 1986) (equation (4) and Figure 2.4), where LDT = background turbidity dichroism,α = constant, λ = wavelength (nm), k= turbidity constant.
( )
T k
-0.001 -0.0005 0 0.0005 0.001 0.0015 0.002 0.0025 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 Wavlength (nm) L D (1 0 -3 ab so rb an ce u n it s) Spectrum Scattering fit Corrected spectrum
Figure 2.4 Linear dichroism light scattering correction.The method given in (Nordhet al., 1986) (equation (4)) was used to calculate the light scattering due to the liposomes, which was subtracted from the spectrum.
2.14 .6 Attenuated to tal reflectan ce Fou ri er transfo rm infrared spectro scopy (ATR-FT IR)
Liposomes with a 30:1 lipid to peptide ratio were prepared with dH2O (§2.13.6), with
a final lipid concentration of 10 mg/mL. A Bruker Tensor spectrometer was used containing a high-sensitivity MCT/A detector cooled with liquid nitrogen. Water vapour and carbon dioxide were evacuated from the machine by purging using dry compressed air from an external source. The sample was deposited onto a trapezoidal germanium internal reflection element and bulk water was removed using a diffused stream of nitrogen. 1000 interferograms were recorded and their average was used for analysis. For deuterium exchange, deuterium saturated nitrogen gas was passed over the sample for 22 hours and a spectrum was recorded. To obtain information on the secondary structure of the peptide the spectra were analysed in the 1600–1700 cm-1 region, which corresponds to the amide I peak. The band-narrowing technique of Fourier self-deconvolution was applied as well as a baseline correction using the OMNIC spectra software (Thermo Scientific). Gaussian curves were fitted to the resulting spectra using GRAMS software (Thermo Scientific).
2.14 .7 Intrinsi c fluo res cen ce
Samples with lipid were prepared as for LD (§2.14.5). Emission spectra were taken on a Perkin Elmer LS50B fluorimeter exciting at 280 nm and recording between 300 and 400 nm, with an excitation slit of 2.5, an emission slit of 2.5 and a scan speed of 50. Spectra of peptide in TFE were also recorded. The fluorescence spectra of the peptide in different environments were determined by subtracting spectra of the buffer without peptide.