• According to FRET, DLS, AUC, and QENS, it seems likely, although not finally established,
that the size of these particles does not vary between the CAC and 240 gÁL
À1
, that is, over five
orders of magnitude (Gohon et al. 2006; Giusti et al. 2012; Tehei et al. 2014). Small particles
thus seem to occupy a very large region of the phase diagram of A8-35, which is of great
practical interest for experiments where MP/APol complexes must be highly concentrated,
such as for EINS and QENS measurements (Tehei et al. 2014), in crystallization attempts
(Charvolin et al. 2014) (Chap. 11), in solid-state NMR, or for certain types of light spectroscopy experiments (see e.g. Polovinkin et al. 2014; Chap. 8, § 8.2.2).
• At pH ! 7.0, most carboxylic moieties are deprotonated (Gohon et al. 2004).
• The mass of the particles does not change in the pH range 6.8–9.2 nor between 5 and 20
C
(Gohon et al. 2006).
• A moderate repulsion between particles is observed in the presence of 100 mM NaCl, the
second virial coefficient, A 2 , increasing from ~15 to ~50 mLÁg
À1 between pH 6.8 and pH 9.2
(Gohon et al. 2006).
• Judging from the results of SAXS measurements carried out on MP/A8-35 complexes, the
repulsion between particles probably vanishes between 300 and 500 mM NaCl, above which
an attractive regime develops (Popot et al. 2003) (cf. Fig. 11.13 in Chap. 11).
• According to SANS data, an attractive regime also develops, in the presence of 100 mM
NaCl, upon addition of 5–10% 4-kDa polyethylene glycol to a 20-gÁL
À1 solution of MP/A835 complexes (Charvolin et al. 2014).
• Lowering the pH to 7 induces aggregation (Gohon et al. 2006).
• Multivalent ions induce aggregation (Picard et al. 2006).
• Given that MP/A8-35 complexes can be frozen without inactivating the protein nor causing it
to aggregate (Gohon et al. 2008), it seems probable that the APol belt and, by extension,
A8-35 particles are not strongly perturbed by freezing.
4.3.2
Solution Properties of Other Amphipols
A8-35 is the only APol whose solution properties have been extensively studied by such a broad
variety of complementary methods. Data for other APols are most often limited to SEC or DLS
estimates of the size of the particles they form in aqueous solutions, except for glucose-based NAPols
and SMALPs (see Table 4.4). Table 4.4 also indicates which of the listed APols are (or can be
expected to be) either sensitive or largely insensitive to aggregation in the presence of Ca
2+ ions or at
acidic pH.
A8-75 and SAPols are two PAA-based APols with the same backbone length distribution and
extent of grafting with octylamine as A8-35 (Fig. 4.1). However, they lack isopropylamine, the
corresponding carboxylates being either left free (A8-75) or grafted with taurine (SAPols), resulting
in a much higher charge density. The particles they form comigrate with A8-35 particles upon SEC
(Dahmane et al. 2011) (Fig. 4.26A, B), suggesting, but not demonstrating, a similar size, mass, and size
distribution (because the higher charge density may speed up elution, the particles may actually be
somewhat smaller than those of A8-35). SAPol particles are insensitive to pH (Dahmane et al. 2011)
(Fig. 4.26B) and largely insensitive to the presence of Ca
2+ or Mg
2+ ions (Picard et al. 2006)
(Fig. 4.26D).
“Blocky” copolymers similar to A8-75 but derived from poly(methacrylic acid) and in which the
octyl groups are distributed in a nonrandom manner have been observed to assemble into markedly
192
4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols
that the size of these particles does not vary between the CAC and 240 gÁL
À1
, that is, over five
orders of magnitude (Gohon et al. 2006; Giusti et al. 2012; Tehei et al. 2014). Small particles
thus seem to occupy a very large region of the phase diagram of A8-35, which is of great
practical interest for experiments where MP/APol complexes must be highly concentrated,
such as for EINS and QENS measurements (Tehei et al. 2014), in crystallization attempts
(Charvolin et al. 2014) (Chap. 11), in solid-state NMR, or for certain types of light spectroscopy experiments (see e.g. Polovinkin et al. 2014; Chap. 8, § 8.2.2).
• At pH ! 7.0, most carboxylic moieties are deprotonated (Gohon et al. 2004).
• The mass of the particles does not change in the pH range 6.8–9.2 nor between 5 and 20
C
(Gohon et al. 2006).
• A moderate repulsion between particles is observed in the presence of 100 mM NaCl, the
second virial coefficient, A 2 , increasing from ~15 to ~50 mLÁg
À1 between pH 6.8 and pH 9.2
(Gohon et al. 2006).
• Judging from the results of SAXS measurements carried out on MP/A8-35 complexes, the
repulsion between particles probably vanishes between 300 and 500 mM NaCl, above which
an attractive regime develops (Popot et al. 2003) (cf. Fig. 11.13 in Chap. 11).
• According to SANS data, an attractive regime also develops, in the presence of 100 mM
NaCl, upon addition of 5–10% 4-kDa polyethylene glycol to a 20-gÁL
À1 solution of MP/A835 complexes (Charvolin et al. 2014).
• Lowering the pH to 7 induces aggregation (Gohon et al. 2006).
• Multivalent ions induce aggregation (Picard et al. 2006).
• Given that MP/A8-35 complexes can be frozen without inactivating the protein nor causing it
to aggregate (Gohon et al. 2008), it seems probable that the APol belt and, by extension,
A8-35 particles are not strongly perturbed by freezing.
4.3.2
Solution Properties of Other Amphipols
A8-35 is the only APol whose solution properties have been extensively studied by such a broad
variety of complementary methods. Data for other APols are most often limited to SEC or DLS
estimates of the size of the particles they form in aqueous solutions, except for glucose-based NAPols
and SMALPs (see Table 4.4). Table 4.4 also indicates which of the listed APols are (or can be
expected to be) either sensitive or largely insensitive to aggregation in the presence of Ca
2+ ions or at
acidic pH.
A8-75 and SAPols are two PAA-based APols with the same backbone length distribution and
extent of grafting with octylamine as A8-35 (Fig. 4.1). However, they lack isopropylamine, the
corresponding carboxylates being either left free (A8-75) or grafted with taurine (SAPols), resulting
in a much higher charge density. The particles they form comigrate with A8-35 particles upon SEC
(Dahmane et al. 2011) (Fig. 4.26A, B), suggesting, but not demonstrating, a similar size, mass, and size
distribution (because the higher charge density may speed up elution, the particles may actually be
somewhat smaller than those of A8-35). SAPol particles are insensitive to pH (Dahmane et al. 2011)
(Fig. 4.26B) and largely insensitive to the presence of Ca
2+ or Mg
2+ ions (Picard et al. 2006)
(Fig. 4.26D).
“Blocky” copolymers similar to A8-75 but derived from poly(methacrylic acid) and in which the
octyl groups are distributed in a nonrandom manner have been observed to assemble into markedly
192
4 Chemical Structure, Synthesis, and Physical-Chemical Properties of Amphipols