peptide is on the membrane surface, in the head group region, or
buried. When a ligand or other peptide/protein binds to a membrane protein, additional LD signals should be apparent. Liposome
flow LD experiments can be used to follow the kinetics of membrane binding if it happens slowly enough. Normal Couette flow
cells have a deadtime of about 30 s. Rapid injection [15] and
channel flow systems [16] can be used to reduce the dead time to
500 μs and 25 μs, respectively, though with the penalty of more
sample and worse signal-to-noise relative to Couette flow.
One of the challenges of liposome LD spectroscopy is that
overlaid on any liposome LD spectrum, one collects a scattering
contribution as the unilamellar lipid vesicles typically used in LD
experiments are of the order of the wavelength of light. Since a
spectrometer detector simply counts the photons that are incident
onto it, photons that do not arrive due to scattering are not
differentiated from those missing due to absorbance. The spectrum
in Fig. 2b shows the original liposome flow LD spectrum of diphenyl hexatriene (DPH) and after correction for scattering.
Overall flow LD is the ideal technique for determining the
orientation of different chromophores with respect to the membrane normal. Some examples have been given above. To illustrate
the method, further examples of antimicrobial peptides, aurein 2.5
and gramicidin are considered below as well as another common
chromophore, fluorene, often used to increase the hydrophobicity
and hence membrane binding of peptides. How LD can be used
both for geometry, structure analysis and probing kinetic processes
is considered. Kinetic analysis usually involves identifying binding
(appearance of an LD signal), insertion (sign change), often followed by loss of signal if the inserted protein or peptide disrupts the
membrane [17]. One of the great advantages of LD is that only
oriented molecules give a signal, so peptides that are not membrane
bound are invisible. Increased sensitivity and selectivity is sometimes possible by undertaking fluorescence detected LD [12, 18].
2 Materials
All solutions should be prepared using ultrapure water (18 MΩ-cm
at 25
C) and analytical grade reagents where available. Follow all
waste disposal regulations when disposing of materials. The choice
of lipid for any experiment is complex. In our experience, the short
lipid DMPC (Fig. 5) forms the least stable liposomes and POPC or
naturally occurring mixtures are easier to use.
454
Matthew R. Hicks et al.
buried. When a ligand or other peptide/protein binds to a membrane protein, additional LD signals should be apparent. Liposome
flow LD experiments can be used to follow the kinetics of membrane binding if it happens slowly enough. Normal Couette flow
cells have a deadtime of about 30 s. Rapid injection [15] and
channel flow systems [16] can be used to reduce the dead time to
500 μs and 25 μs, respectively, though with the penalty of more
sample and worse signal-to-noise relative to Couette flow.
One of the challenges of liposome LD spectroscopy is that
overlaid on any liposome LD spectrum, one collects a scattering
contribution as the unilamellar lipid vesicles typically used in LD
experiments are of the order of the wavelength of light. Since a
spectrometer detector simply counts the photons that are incident
onto it, photons that do not arrive due to scattering are not
differentiated from those missing due to absorbance. The spectrum
in Fig. 2b shows the original liposome flow LD spectrum of diphenyl hexatriene (DPH) and after correction for scattering.
Overall flow LD is the ideal technique for determining the
orientation of different chromophores with respect to the membrane normal. Some examples have been given above. To illustrate
the method, further examples of antimicrobial peptides, aurein 2.5
and gramicidin are considered below as well as another common
chromophore, fluorene, often used to increase the hydrophobicity
and hence membrane binding of peptides. How LD can be used
both for geometry, structure analysis and probing kinetic processes
is considered. Kinetic analysis usually involves identifying binding
(appearance of an LD signal), insertion (sign change), often followed by loss of signal if the inserted protein or peptide disrupts the
membrane [17]. One of the great advantages of LD is that only
oriented molecules give a signal, so peptides that are not membrane
bound are invisible. Increased sensitivity and selectivity is sometimes possible by undertaking fluorescence detected LD [12, 18].
2 Materials
All solutions should be prepared using ultrapure water (18 MΩ-cm
at 25
C) and analytical grade reagents where available. Follow all
waste disposal regulations when disposing of materials. The choice
of lipid for any experiment is complex. In our experience, the short
lipid DMPC (Fig. 5) forms the least stable liposomes and POPC or
naturally occurring mixtures are easier to use.
454
Matthew R. Hicks et al.