has 22 TM helices. On the basis of the above calculations, one would expect it to bind ~96 kDa A8-35.
The experimental estimate is only 49–63 kDa (Popot et al. 2003; Charvolin et al. 2014). Note also that
it is not unreasonable to expect that the ionic strength may affect the volume and mass of the belts of
ionic APols, because it modulates the repulsion between charged polar groups. Despite these
uncertainties, estimating a priori the probable mass ratio of MP to APol in complexes is useful to
provide guidelines when planning trapping experiments, or when undertaking to measure experimentally the amount of bound APol.
5.9.2.3 How to Experimentally Measure the Quantity of APols Bound per MP?
As previously mentioned, the mass of APol to add for trapping is in excess of that of APol that
actually binds to the surface of the MP. After trapping, some APol remains present as free particles in
the sample. Measuring the amount of bound APols can be carried out by several approaches. Initial
experiments resorted to radioactively labeled APols (see e.g. Tribet et al. 1997; Gohon et al. 2008, and
Chap. 9, Fig. 9.3). Later analyses were facilitated by using FAPols as tracers.
• Prepare a stock solution of APol/FAPol mixture.
Several FAPols carrying various fluorescent probes are available (see Chap. 4, Table 4.5). The
choice of FAPol depends on the absorbance spectrum of the protein of interest. For instance, if the
protein absorbs only at 280 nm, FAPol NBD , which shows a maximum absorbance at 490 nm, is
suitable. On the other hand, if the protein also absorbs visible light, as BR does, another FAPol, like
FAPol AF657 , may be chosen in order to avoid an overlap between the protein and FAPol absorbance
bands.
Pure FAPols usually absorb too much at the peak of absorbance of the fluorophore, and possibly
also at 280 nm, interfering with protein determination. They are better used diluted with
nonfluorescent APol (A8-35). Because labeled and unlabeled APols mix freely and rapidly in salty
aqueous solutions (Zoonens et al. 2007), they can be mixed from two stock solutions prepared at
100 gÁL
À1 . A convenient FAPol/A8-35 ratio is one at which the absorbance of the FAPol at its maximal
absorbance wavelength is, in the complexes with the MP, ~25% of that of the protein at 280 nm. This
ratio can be estimated a priori based on the extinction coefficients of the protein and FAPol, the
estimate of the amount of APols bound per MP (cf. § 5.9.3.2), and the MP/APol mass ratio needed for
trapping. If the protein possesses many tryptophan residues, its extinction coefficient may be high
enough so that no dilution of the FAPol stock solution is necessary.
• Measure the spectral absorbance of the FAPol/A8-35 mixture at 10 gÁL
À1 if FAPol and A8-35
were mixed in a 1:9 ratio (or pure FAPol at 1 gÁL
À1 if dilution with A8-35 is not necessary).
Determine the relative contribution of APols at 280 nm and at the peak of absorbance. (Note:
even if neither APol nor FAPol absorbs significantly at 280 nm, it is advisable to check on it.)
After the complexes have been formed, they must be separated from the excess APol used at the
trapping step for the MP/APol ratio in the complexes to be estimated. As of today, three different
separation methods have been resorted to.
Method 1. Size Exclusion Chromatography (SEC)
This approach is appropriate for MPs that are large enough – !40 kDa, say – for MP/APol
complexes and free APol particles to be sufficiently resolved.
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5 Formation and Properties of Membrane Protein/Amphipol Complexes
The experimental estimate is only 49–63 kDa (Popot et al. 2003; Charvolin et al. 2014). Note also that
it is not unreasonable to expect that the ionic strength may affect the volume and mass of the belts of
ionic APols, because it modulates the repulsion between charged polar groups. Despite these
uncertainties, estimating a priori the probable mass ratio of MP to APol in complexes is useful to
provide guidelines when planning trapping experiments, or when undertaking to measure experimentally the amount of bound APol.
5.9.2.3 How to Experimentally Measure the Quantity of APols Bound per MP?
As previously mentioned, the mass of APol to add for trapping is in excess of that of APol that
actually binds to the surface of the MP. After trapping, some APol remains present as free particles in
the sample. Measuring the amount of bound APols can be carried out by several approaches. Initial
experiments resorted to radioactively labeled APols (see e.g. Tribet et al. 1997; Gohon et al. 2008, and
Chap. 9, Fig. 9.3). Later analyses were facilitated by using FAPols as tracers.
• Prepare a stock solution of APol/FAPol mixture.
Several FAPols carrying various fluorescent probes are available (see Chap. 4, Table 4.5). The
choice of FAPol depends on the absorbance spectrum of the protein of interest. For instance, if the
protein absorbs only at 280 nm, FAPol NBD , which shows a maximum absorbance at 490 nm, is
suitable. On the other hand, if the protein also absorbs visible light, as BR does, another FAPol, like
FAPol AF657 , may be chosen in order to avoid an overlap between the protein and FAPol absorbance
bands.
Pure FAPols usually absorb too much at the peak of absorbance of the fluorophore, and possibly
also at 280 nm, interfering with protein determination. They are better used diluted with
nonfluorescent APol (A8-35). Because labeled and unlabeled APols mix freely and rapidly in salty
aqueous solutions (Zoonens et al. 2007), they can be mixed from two stock solutions prepared at
100 gÁL
À1 . A convenient FAPol/A8-35 ratio is one at which the absorbance of the FAPol at its maximal
absorbance wavelength is, in the complexes with the MP, ~25% of that of the protein at 280 nm. This
ratio can be estimated a priori based on the extinction coefficients of the protein and FAPol, the
estimate of the amount of APols bound per MP (cf. § 5.9.3.2), and the MP/APol mass ratio needed for
trapping. If the protein possesses many tryptophan residues, its extinction coefficient may be high
enough so that no dilution of the FAPol stock solution is necessary.
• Measure the spectral absorbance of the FAPol/A8-35 mixture at 10 gÁL
À1 if FAPol and A8-35
were mixed in a 1:9 ratio (or pure FAPol at 1 gÁL
À1 if dilution with A8-35 is not necessary).
Determine the relative contribution of APols at 280 nm and at the peak of absorbance. (Note:
even if neither APol nor FAPol absorbs significantly at 280 nm, it is advisable to check on it.)
After the complexes have been formed, they must be separated from the excess APol used at the
trapping step for the MP/APol ratio in the complexes to be estimated. As of today, three different
separation methods have been resorted to.
Method 1. Size Exclusion Chromatography (SEC)
This approach is appropriate for MPs that are large enough – !40 kDa, say – for MP/APol
complexes and free APol particles to be sufficiently resolved.
316
5 Formation and Properties of Membrane Protein/Amphipol Complexes