by any APol under a functional form and bind their ligands with the same specificity and affinity as
measured in a membrane environment. However, A8-35 is observed to inhibit or slow down G protein
activation and arrestin recruitment, whereas NAPols and SMA do not. The difference between A8-35
and NAPols may probably be attributed to the polyanionic character of A8-35 vs. the non-ionic one of
NAPols. It is however surprising, if electrostatic interactions are involved, that the equally polyanionic
SMA be not inhibitory as well (Logez et al. 2016). A possible interpretation is that more abundant
lipids in SMALPs keep the polymer further away from the receptor.
As a rule, no interference is observed with the binding of small water-soluble ligands to APoltrapped MPs, such as that of Ca
2+ and ATP to SERCA1a (Champeil et al. 2000), that of small
acetylcholine analogs to the nAChR (Martinez et al. 2002; Charvolin et al. 2009), or that of various
ligands to GPCRs (Dahmane et al. 2009; Catoire et al. 2010a, 2011; Banères et al. 2011; Bazzacco
et al. 2012; Rahmeh et al. 2012). Two GPCRs expressed in vitro in the presence of NVoy, CRFR1, and
CRFR2β have been shown to bind their ligands (Klammt et al. 2011), as well as three SMA-extracted
GPCRs, the adenosine A 2A receptor (A 2A R), ghrelin GHS-R1a receptor, and melatonin MT1R
receptor (Jamshad et al. 2015a, b; Logez et al. 2016). It is to be noted that A8-35 does not interfere
with the binding of leukotriene LTB 4 to the BLT1 or BLT2 receptors (Dahmane et al. 2009; Catoire
et al. 2010a), even though, given the hydrophobicity of LTB 4 , its binding site must be itself quite
hydrophobic and could in principle attract APol octyl chains. APols do not block either the binding of
large water-soluble partners, such as that of α-bungarotoxin (α-Bgt) (8 kDa) to the nAChR (Charvolin
et al. 2009), that of bacteriophage T5 protein pb5 (68 kDa) to FhuA (Basit et al. 2012), nor the
recognition of several MP targets by synthetic proteins called αReps (15–20 kDa) (Ferrandez et al.
2014; see Chap. 13, § 13.2.3) or by antibodies (~150 kDa) (Charvolin et al. 2009; Tifrea et al. 2011;
Le Bon et al. 2014), nor, as mentioned above, the interaction of GPCRs with G proteins and arrestin
(Bazzacco et al. 2012; Rahmeh et al. 2012). In the latter case, however, it has been observed, as
mentioned above, that interactions are less efficient with A8-35-trapped than with NAPol-trapped
GPCRs. Also, specific binding of cationic ligands can be difficult to measure in the presence of A8-35,
because of a high background of non-specific binding (Ferrandez et al. 2014; Mary et al. 2014, and
unpublished data by various groups). Experiments in which retinal was added to bacterio-opsin
(BO) refolded in the presence of A8-35 indicate that this very hydrophobic ligand can be delivered
a posteriori to the apoprotein (Dahmane et al. 2013). Retinal presumably moves from free APol
particles, where it must partition, to the protein-bound APol belt during collisions between the particles
and BO/APol complexes and then inserts itself into the α-helix bundle. The covalent Schiff base then
forms spontaneously (Huang et al. 1981).
Many MPs exhibit enzymatic activity after trapping with APols (Table 5.1). Thus, E. coli
diacylglycerol kinase (DAGK) retains full enzymatic activity upon transfer from decylmaltoside to
PMAL-B-100 (Gorzelle et al. 2002). The bacterial outer membrane enzyme PagP retains phospholipase activity after being trapped in SMALPs (Knowles et al. 2009), as well as in A8-35, A8-75,
A34-35, A34-75, SAPols, and NAPols (Leney et al. 2012; Calabrese et al. 2015; Watkinson et al.
2015). The transmembrane domain of the bacterial EII
mtl mannitol permease performs the transphosphorylation from phosphoenolpyruvate to mannitol more rapidly after trapping in A8-35 than it does
in detergent solution (Opačić et al. 2014b). Similarly, the basal ATPase activity of ABCA4, a
photoreceptor-specific ABC transporter, is higher after trapping with A8-35 than in detergent solution
(Tsybovsky et al. 2013). Cytochrome bc 1 transfers electrons from ubiquinol to oxidized cytochrome
c at comparable rates whether solubilized in DDM or trapped by A8-35 (Charvolin et al. 2014). Other
enzymatic reactions that have been evidenced include the protease activity of OmpT in A8-35, A8-75,
A34-35, A34-75, SAPols, and NAPols (Leney et al. 2012; Calabrese et al. 2015; Watkinson et al.
2015), and the autophosphorylation of SMA-trapped E. coli tyrosine kinase (Li et al. 2015a).
Nucleotide binding by several SMA-trapped eukaryotic ABC transporters could be demonstrated,
5.4 Functionality of Amphipol-Trapped Membrane Proteins
289
measured in a membrane environment. However, A8-35 is observed to inhibit or slow down G protein
activation and arrestin recruitment, whereas NAPols and SMA do not. The difference between A8-35
and NAPols may probably be attributed to the polyanionic character of A8-35 vs. the non-ionic one of
NAPols. It is however surprising, if electrostatic interactions are involved, that the equally polyanionic
SMA be not inhibitory as well (Logez et al. 2016). A possible interpretation is that more abundant
lipids in SMALPs keep the polymer further away from the receptor.
As a rule, no interference is observed with the binding of small water-soluble ligands to APoltrapped MPs, such as that of Ca
2+ and ATP to SERCA1a (Champeil et al. 2000), that of small
acetylcholine analogs to the nAChR (Martinez et al. 2002; Charvolin et al. 2009), or that of various
ligands to GPCRs (Dahmane et al. 2009; Catoire et al. 2010a, 2011; Banères et al. 2011; Bazzacco
et al. 2012; Rahmeh et al. 2012). Two GPCRs expressed in vitro in the presence of NVoy, CRFR1, and
CRFR2β have been shown to bind their ligands (Klammt et al. 2011), as well as three SMA-extracted
GPCRs, the adenosine A 2A receptor (A 2A R), ghrelin GHS-R1a receptor, and melatonin MT1R
receptor (Jamshad et al. 2015a, b; Logez et al. 2016). It is to be noted that A8-35 does not interfere
with the binding of leukotriene LTB 4 to the BLT1 or BLT2 receptors (Dahmane et al. 2009; Catoire
et al. 2010a), even though, given the hydrophobicity of LTB 4 , its binding site must be itself quite
hydrophobic and could in principle attract APol octyl chains. APols do not block either the binding of
large water-soluble partners, such as that of α-bungarotoxin (α-Bgt) (8 kDa) to the nAChR (Charvolin
et al. 2009), that of bacteriophage T5 protein pb5 (68 kDa) to FhuA (Basit et al. 2012), nor the
recognition of several MP targets by synthetic proteins called αReps (15–20 kDa) (Ferrandez et al.
2014; see Chap. 13, § 13.2.3) or by antibodies (~150 kDa) (Charvolin et al. 2009; Tifrea et al. 2011;
Le Bon et al. 2014), nor, as mentioned above, the interaction of GPCRs with G proteins and arrestin
(Bazzacco et al. 2012; Rahmeh et al. 2012). In the latter case, however, it has been observed, as
mentioned above, that interactions are less efficient with A8-35-trapped than with NAPol-trapped
GPCRs. Also, specific binding of cationic ligands can be difficult to measure in the presence of A8-35,
because of a high background of non-specific binding (Ferrandez et al. 2014; Mary et al. 2014, and
unpublished data by various groups). Experiments in which retinal was added to bacterio-opsin
(BO) refolded in the presence of A8-35 indicate that this very hydrophobic ligand can be delivered
a posteriori to the apoprotein (Dahmane et al. 2013). Retinal presumably moves from free APol
particles, where it must partition, to the protein-bound APol belt during collisions between the particles
and BO/APol complexes and then inserts itself into the α-helix bundle. The covalent Schiff base then
forms spontaneously (Huang et al. 1981).
Many MPs exhibit enzymatic activity after trapping with APols (Table 5.1). Thus, E. coli
diacylglycerol kinase (DAGK) retains full enzymatic activity upon transfer from decylmaltoside to
PMAL-B-100 (Gorzelle et al. 2002). The bacterial outer membrane enzyme PagP retains phospholipase activity after being trapped in SMALPs (Knowles et al. 2009), as well as in A8-35, A8-75,
A34-35, A34-75, SAPols, and NAPols (Leney et al. 2012; Calabrese et al. 2015; Watkinson et al.
2015). The transmembrane domain of the bacterial EII
mtl mannitol permease performs the transphosphorylation from phosphoenolpyruvate to mannitol more rapidly after trapping in A8-35 than it does
in detergent solution (Opačić et al. 2014b). Similarly, the basal ATPase activity of ABCA4, a
photoreceptor-specific ABC transporter, is higher after trapping with A8-35 than in detergent solution
(Tsybovsky et al. 2013). Cytochrome bc 1 transfers electrons from ubiquinol to oxidized cytochrome
c at comparable rates whether solubilized in DDM or trapped by A8-35 (Charvolin et al. 2014). Other
enzymatic reactions that have been evidenced include the protease activity of OmpT in A8-35, A8-75,
A34-35, A34-75, SAPols, and NAPols (Leney et al. 2012; Calabrese et al. 2015; Watkinson et al.
2015), and the autophosphorylation of SMA-trapped E. coli tyrosine kinase (Li et al. 2015a).
Nucleotide binding by several SMA-trapped eukaryotic ABC transporters could be demonstrated,
5.4 Functionality of Amphipol-Trapped Membrane Proteins
289