9.5.1.4 Compiling the Parameters That Will Be Required for the Analysis
The partial specific volumes
v, in mL‧g
À1 ; the extinction coefficients ε, in L‧g
À1
‧cm
À1 , at the
wavelengths of measurement in the AUC; the refractive index increments (∂n/∂c) of the components
(protein and APol); the protein molar mass from its sequence; and the buffer density and viscosity
should be known. Complementary information on the hydrodynamic radius of the complex, possible
protein association states, amount of bound lipids, etc. should be compiled.
• Parameters for the protein can be calculated from its sequence, using, for example, the
program Sedfit (http://www.analyticalultracentrifugation.com).
If the protein is glycosylated, glycosylation has to be taken into account (see e.g. le Maire
et al. 2008).
• Parameters for the APol. The specific volume
v of the sodium salt of A8-35 is 0.809 mLÁg
À1 ,
its density, ρ ¼ 1= v ¼ 1:236 g Á mL
À1 . However, because of the polyelectrolyte nature of
A8-35, the apparent specific volume to be considered in dilute salt solution is 0.866 mLÁg
À1
(Gohon et al. 2004, 2006). (∂n/∂c) ¼ 0.15 g‧mL
À1 was used as a likely value for A8-35
(Gohon et al. 2008). It can be experimentally measured, as well as the extinction coefficient,
from SV experiments with APol samples of known concentrations in the g‧L
À1 range
(cf. Salvay and Ebel 2006).
• Parameters for the buffer can be obtained from tabulated data, e.g. with the program
SEDNTERP (http://www.jphilo.mailway.com) for usual or simple buffers, or measured
with a viscometer and a densimeter.
9.5.1.5 Running SV Experiments
The parameters to be set are the temperature, the angular velocity, the interval between scans, and the
total time of sedimentation.
• The temperature can be chosen between 4 and 30
C.
• Angular velocities of 42,000 or 50,000 rpm (130,000 or 184,000 Â g) are usually equally
convenient. For very large complexes (above 500 kDa), the angular velocity should be
decreased to, for example 35,000 rpm (90,000 Â g).
• Interval between scans: fix 5-min intervals. The effective interval will be 5 min or larger,
depending on the number of cells and number of wavelengths; on the XLI AUC, the interval
between scans is 12–15 min for four cells measured at two wavelengths and with interference
optics.
• Total duration: we typically request the acquisition of 999 scans (the maximum possible
input) and run the AUC experiment overnight before stopping it manually after, typically,
100 scans. The relevant scans (before everything is pelleted) will be selected for the analysis:
for instance, small MPs, like BR or tOmpA, will be pelleted in ~7 h.
9.5.1.6 c(s) Analysis
Analyze the SV profiles for each detection mode (different wavelengths and interference optics) with the
c(s) analysis model of the program Sedfit (http://www.analyticalultracentrifugation.com) (for details, see
Gohon et al. 2008). The distribution c(s) of sedimentation coefficients (s) shows peaks reflecting the
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9 Solution Studies of Membrane Protein/Amphipol Complexes
The partial specific volumes
v, in mL‧g
À1 ; the extinction coefficients ε, in L‧g
À1
‧cm
À1 , at the
wavelengths of measurement in the AUC; the refractive index increments (∂n/∂c) of the components
(protein and APol); the protein molar mass from its sequence; and the buffer density and viscosity
should be known. Complementary information on the hydrodynamic radius of the complex, possible
protein association states, amount of bound lipids, etc. should be compiled.
• Parameters for the protein can be calculated from its sequence, using, for example, the
program Sedfit (http://www.analyticalultracentrifugation.com).
If the protein is glycosylated, glycosylation has to be taken into account (see e.g. le Maire
et al. 2008).
• Parameters for the APol. The specific volume
v of the sodium salt of A8-35 is 0.809 mLÁg
À1 ,
its density, ρ ¼ 1= v ¼ 1:236 g Á mL
À1 . However, because of the polyelectrolyte nature of
A8-35, the apparent specific volume to be considered in dilute salt solution is 0.866 mLÁg
À1
(Gohon et al. 2004, 2006). (∂n/∂c) ¼ 0.15 g‧mL
À1 was used as a likely value for A8-35
(Gohon et al. 2008). It can be experimentally measured, as well as the extinction coefficient,
from SV experiments with APol samples of known concentrations in the g‧L
À1 range
(cf. Salvay and Ebel 2006).
• Parameters for the buffer can be obtained from tabulated data, e.g. with the program
SEDNTERP (http://www.jphilo.mailway.com) for usual or simple buffers, or measured
with a viscometer and a densimeter.
9.5.1.5 Running SV Experiments
The parameters to be set are the temperature, the angular velocity, the interval between scans, and the
total time of sedimentation.
• The temperature can be chosen between 4 and 30
C.
• Angular velocities of 42,000 or 50,000 rpm (130,000 or 184,000 Â g) are usually equally
convenient. For very large complexes (above 500 kDa), the angular velocity should be
decreased to, for example 35,000 rpm (90,000 Â g).
• Interval between scans: fix 5-min intervals. The effective interval will be 5 min or larger,
depending on the number of cells and number of wavelengths; on the XLI AUC, the interval
between scans is 12–15 min for four cells measured at two wavelengths and with interference
optics.
• Total duration: we typically request the acquisition of 999 scans (the maximum possible
input) and run the AUC experiment overnight before stopping it manually after, typically,
100 scans. The relevant scans (before everything is pelleted) will be selected for the analysis:
for instance, small MPs, like BR or tOmpA, will be pelleted in ~7 h.
9.5.1.6 c(s) Analysis
Analyze the SV profiles for each detection mode (different wavelengths and interference optics) with the
c(s) analysis model of the program Sedfit (http://www.analyticalultracentrifugation.com) (for details, see
Gohon et al. 2008). The distribution c(s) of sedimentation coefficients (s) shows peaks reflecting the
442
9 Solution Studies of Membrane Protein/Amphipol Complexes