surfactant-free buffer results in MP aggregation, which is certainly accompanied by some APol
desorption, part of the original MP/APol contacts being replaced by MP/MP ones (§ 5.2.1). If,
however, a biotinylated MP trapped in APol is immobilized onto an avidin-coated surface plasmon
resonance (SPR) chip and the chip extensively washed with surfactant-free buffer, no desorption is
observed (ibid., Fig. 5.7). Under such conditions, the association between the protein and the polymer
is permanent, even though it is not covalent. Because APols can be easily functionalized (Chap. 4,
§ 4.4), they provide a way to indirectly functionalize any MP they bind to, without the protein itself
having to be chemically or genetically modified. This observation is at the basis of a great many
applications. In the present chapter, we will describe those tagged APols that have been thoroughly
characterized or are under development and consider those applications that have already been
validated or represent likely prospects.
In the past few decades, protein microarrays, protein sensor chips, and microbeads have emerged
as tools of choice for obtaining information about protein functions and interactions (see e.g. Weinrich
et al. 2009). Protein arrays combined with sensitive optical detection systems such as SPR or
fluorescence microscopy give access to multiplexing and high-throughput analyses, which have a
high potential for screening studies. These techniques can also provide important information regarding protein affinities and kinetics of interaction using minimal amounts of reagents. All surface-based
assays however require that the target proteins be immobilized under such conditions as to preserve
their native structure and biological activity (see e.g. Jonkheijm et al. 2008). Functional immobilization
of MPs can be achieved by covering bare or modified surfaces with native (Yang and Lundahl 1994;
Gottschalk et al. 2000; Martinez et al. 2003; Perez et al. 2006) or reconstituted (Bieri et al. 1999; Früh
et al. 2011) membrane fragments. These methods have the advantage of allowing the investigation of
MPs in their native lipid environment, but they suffer from high levels of non-specific binding and
from a relatively unstable immobilization, which limit their applications (Früh et al. 2011). More stable
immobilization can be achieved by fusing genetically or chemically small peptide or protein tags to the
target MPs for mediating reversible or irreversible immobilization of MP/detergent complexes
(Schmid et al. 1998; Friedrich et al. 2004; Harding et al. 2006). These strategies require a covalent
modification of the MPs under study, and they suffer from technical hurdles due to the use of
detergents. APols offer a highly versatile alternative for MP immobilization. Their advantages include
the following:
(i) No modification of the target MP is required.
(ii) APol-trapped MPs are, very generally, stabilized as compared to handling them in detergent
solutions (Chap. 5, § 5.5).
(iii) The same APol can be used for any number of MPs, or mixtures of MPs, greatly simplifying
the chemistry work.
(iv) Using mixtures of APols, MPs can be simultaneously tagged and labeled, e.g. with fluorescent APols, so as to visualize the immobilization process.
(v) Analyses can be conducted in detergent-free solutions.
At the time of this writing, more than 20 labeled or functionalized APols have been synthesized
and characterized, including eight tagged ones (see Chap. 4, § 4.4, Table 4.5). Biotin has been attached
to A8-35 (Charvolin et al. 2009), to phosphorylcholine-based APols (Basit et al. 2012), to non-ionic
APols (Ferrandez et al. 2014), and to a styrene-maleic acid copolymer (SMA) (Lindhoud et al. 2016).
A8-35 has also been derivatized with an oligonucleotide (ODN; Le Bon et al. 2014a), with
hexahistidine tags (Giusti et al. 2015), with randomly distributed imidazole moieties (M. Zoonens,
F. Giusti, and C. Le Bon, unpublished results), and with thiomorpholine (F. Giusti, unpublished
results) (see below Table 13.1; for reviews, see Della Pia et al. (2014b), Le Bon et al. (2014b), and
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13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications
desorption, part of the original MP/APol contacts being replaced by MP/MP ones (§ 5.2.1). If,
however, a biotinylated MP trapped in APol is immobilized onto an avidin-coated surface plasmon
resonance (SPR) chip and the chip extensively washed with surfactant-free buffer, no desorption is
observed (ibid., Fig. 5.7). Under such conditions, the association between the protein and the polymer
is permanent, even though it is not covalent. Because APols can be easily functionalized (Chap. 4,
§ 4.4), they provide a way to indirectly functionalize any MP they bind to, without the protein itself
having to be chemically or genetically modified. This observation is at the basis of a great many
applications. In the present chapter, we will describe those tagged APols that have been thoroughly
characterized or are under development and consider those applications that have already been
validated or represent likely prospects.
In the past few decades, protein microarrays, protein sensor chips, and microbeads have emerged
as tools of choice for obtaining information about protein functions and interactions (see e.g. Weinrich
et al. 2009). Protein arrays combined with sensitive optical detection systems such as SPR or
fluorescence microscopy give access to multiplexing and high-throughput analyses, which have a
high potential for screening studies. These techniques can also provide important information regarding protein affinities and kinetics of interaction using minimal amounts of reagents. All surface-based
assays however require that the target proteins be immobilized under such conditions as to preserve
their native structure and biological activity (see e.g. Jonkheijm et al. 2008). Functional immobilization
of MPs can be achieved by covering bare or modified surfaces with native (Yang and Lundahl 1994;
Gottschalk et al. 2000; Martinez et al. 2003; Perez et al. 2006) or reconstituted (Bieri et al. 1999; Früh
et al. 2011) membrane fragments. These methods have the advantage of allowing the investigation of
MPs in their native lipid environment, but they suffer from high levels of non-specific binding and
from a relatively unstable immobilization, which limit their applications (Früh et al. 2011). More stable
immobilization can be achieved by fusing genetically or chemically small peptide or protein tags to the
target MPs for mediating reversible or irreversible immobilization of MP/detergent complexes
(Schmid et al. 1998; Friedrich et al. 2004; Harding et al. 2006). These strategies require a covalent
modification of the MPs under study, and they suffer from technical hurdles due to the use of
detergents. APols offer a highly versatile alternative for MP immobilization. Their advantages include
the following:
(i) No modification of the target MP is required.
(ii) APol-trapped MPs are, very generally, stabilized as compared to handling them in detergent
solutions (Chap. 5, § 5.5).
(iii) The same APol can be used for any number of MPs, or mixtures of MPs, greatly simplifying
the chemistry work.
(iv) Using mixtures of APols, MPs can be simultaneously tagged and labeled, e.g. with fluorescent APols, so as to visualize the immobilization process.
(v) Analyses can be conducted in detergent-free solutions.
At the time of this writing, more than 20 labeled or functionalized APols have been synthesized
and characterized, including eight tagged ones (see Chap. 4, § 4.4, Table 4.5). Biotin has been attached
to A8-35 (Charvolin et al. 2009), to phosphorylcholine-based APols (Basit et al. 2012), to non-ionic
APols (Ferrandez et al. 2014), and to a styrene-maleic acid copolymer (SMA) (Lindhoud et al. 2016).
A8-35 has also been derivatized with an oligonucleotide (ODN; Le Bon et al. 2014a), with
hexahistidine tags (Giusti et al. 2015), with randomly distributed imidazole moieties (M. Zoonens,
F. Giusti, and C. Le Bon, unpublished results), and with thiomorpholine (F. Giusti, unpublished
results) (see below Table 13.1; for reviews, see Della Pia et al. (2014b), Le Bon et al. (2014b), and
592
13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications