should begin 50–100 nm higher in wavelength than the first
sample absorbance signal.
4. A starting set of parameters is 100 nm/min, 1 s response time,
and 1 nm bandwidth or equivalent (see Note 8).
5. Collect a series of LD spectra with the Couette cell stationary,
rotating at about 1000 revolutions per minute, stationary, and
rotating. If the instrument allows it, collect simultaneous LD
and absorbance spectra. Compare the repeat spectra. Any differences are indicative of a kinetic process such as insertion of
the peptide into the membrane or liposome degradation
(Fig. 7).
6. If the Couette cell has the same LD signal at all orientations
(i.e., the quartz is of high quality, see Note 9), then the simplest
baseline is the nonrotating spectrum from step 5. Alternative
baselines are a rotating Couette cell filled with water or filled
with a liposome solution without the analyte.
7. It is often necessary to perform kinetic experiments to see the
time dependence of an insertion process and its consequences
(Fig. 7). As any scattering signal will also change with time, it is
usually necessary to measure full wavelength scans, not single
point measurements, as a function of time.
3.3 Liposome LD
Analysis
3.3.1 Data Processing
1. Begin your data analysis by overlaying plots of your flow LD
sample spectrum with all the baseline options you have collected. In an ideal world, they all overlay outside the absorbance region (thus have the same scattering signal). Choose the
best sample/baseline combination and subtract the baseline
(see Note 10).
2. It may be useful to apply a scattering correction as outlined in
[4] and illustrated in Fig. 2.
3.3.2 Data Interpretation
1. Consider where there are obvious bands in the spectrum and
try to assign them to chromophores of the system. Usually the
lipids can be ignored (see Note 11). α-helices have three accessible transitions that need to be considered and β-sheets have
two with transition polarizations as illustrated in Fig. 3. Peptide
flow LD spectra are often dominated by side chain signals even
in the backbone region (below 240 nm) as aromatic groups are
typically present in higher percentages in membrane-binding
peptides than in a standard protein. Any chromophores such as
the hydrophobic FMOC (fluorenylmethyloxycarbonyl chloride, Fig. 8) added to a peptide to change its properties may
also have significant signals (see Note 12).
2. When the side chain and other chromophore contributions
have been identified, the peptide contributions should be
apparent. Figure 9 illustrates the LD spectra of aurein 2.5
Flow LD of Protein–Membrane Systems
457
sample absorbance signal.
4. A starting set of parameters is 100 nm/min, 1 s response time,
and 1 nm bandwidth or equivalent (see Note 8).
5. Collect a series of LD spectra with the Couette cell stationary,
rotating at about 1000 revolutions per minute, stationary, and
rotating. If the instrument allows it, collect simultaneous LD
and absorbance spectra. Compare the repeat spectra. Any differences are indicative of a kinetic process such as insertion of
the peptide into the membrane or liposome degradation
(Fig. 7).
6. If the Couette cell has the same LD signal at all orientations
(i.e., the quartz is of high quality, see Note 9), then the simplest
baseline is the nonrotating spectrum from step 5. Alternative
baselines are a rotating Couette cell filled with water or filled
with a liposome solution without the analyte.
7. It is often necessary to perform kinetic experiments to see the
time dependence of an insertion process and its consequences
(Fig. 7). As any scattering signal will also change with time, it is
usually necessary to measure full wavelength scans, not single
point measurements, as a function of time.
3.3 Liposome LD
Analysis
3.3.1 Data Processing
1. Begin your data analysis by overlaying plots of your flow LD
sample spectrum with all the baseline options you have collected. In an ideal world, they all overlay outside the absorbance region (thus have the same scattering signal). Choose the
best sample/baseline combination and subtract the baseline
(see Note 10).
2. It may be useful to apply a scattering correction as outlined in
[4] and illustrated in Fig. 2.
3.3.2 Data Interpretation
1. Consider where there are obvious bands in the spectrum and
try to assign them to chromophores of the system. Usually the
lipids can be ignored (see Note 11). α-helices have three accessible transitions that need to be considered and β-sheets have
two with transition polarizations as illustrated in Fig. 3. Peptide
flow LD spectra are often dominated by side chain signals even
in the backbone region (below 240 nm) as aromatic groups are
typically present in higher percentages in membrane-binding
peptides than in a standard protein. Any chromophores such as
the hydrophobic FMOC (fluorenylmethyloxycarbonyl chloride, Fig. 8) added to a peptide to change its properties may
also have significant signals (see Note 12).
2. When the side chain and other chromophore contributions
have been identified, the peptide contributions should be
apparent. Figure 9 illustrates the LD spectra of aurein 2.5
Flow LD of Protein–Membrane Systems
457