such as polyhistidine (Giusti et al. 2015), biotin (Charvolin et al. 2009), or an oligonucleotide (Le Bon
et al. 2014a), all of which have been validated for attaching MPs onto solid beads or chips, could
conceivably be used to associate MPs to fibers for partial orientation in the magnetic field in view of
residual dipolar coupling measurements (see e.g. Prestegard et al. 2000; Bax et al. 2001; Kay 2001;
Torchia 2015, and refs. therein), or to immobilize them onto resins for the purpose of screening banks
of ligands (cf. Früh et al. 2011).
As mentioned at the beginning of this chapter, no report has yet appeared of applying ssNMR to
APol-trapped MPs. This is to some extent logical: ssNMR is particularly well adapted to studying MPs
in their native membrane environment, and little interest may be found in applying it to MPs that have
been extracted from it. Yet, as has been discussed in Chap. 5, there is some evidence that MPs trapped
in APol in the presence of lipids rebind them, forming ternary complexes in which proteins may exhibit
functional properties closer to those observed in native membranes than in detergent solution
(Martinez et al. 2002; Dahmane et al. 2013). Whereas APols themselves are definitely no membrane
mimics, the interactions experienced by MPs trapped in APols in the presence of lipids may resemble
those experienced in the membrane more than one might think when looking at the chemical structure
of APols. Sensitivity is a major issue in ssNMR. APol-trapped BR can be precipitated without
denaturation either by extensive ultracentrifugation (Gohon et al. 2008) or by deliberately aggregating
the complexes, e.g. by adding Ca
2+ ions to A8-35-trapped BR, or multivalent avidin to BR trapped
with a biotinylated APol (Bazzacco 2009). The concentration of BR in the paste thus obtained is much
higher than can be achieved in membrane samples, a highly favorable factor for ssNMR studies.
19 F
ssNMR has been applied to studying the phosphorylation of an E. coli tyrosine kinase incorporated
into SMALPs (Li et al. 2015).
More generally, one may note that there is a dearth of comparative NMR studies of the same MP
in different environments, on the model of those reported in Etzkorn et al. (2013) (§ 10.3.1.2). A very
interesting suggestion is to use data collected in environments closest to the natural one, such as
ssNMR data collected on membrane-integrated MPs or sNMR data collected on ND-trapped ones, to
assess to which extent other surfactants do or do not affect the structure and dynamics of the MP under
study (Howell et al. 2005; Poget and Girvin 2007; Shenkarev et al. 2010; Etzkorn et al. 2013). Such
comparisons would help delineate which perturbations may or may not be expected from one or the
other surfactant, and help to choose, for each experiment, the type of samples that offers the best
compromise between experimental convenience and reliable information.
References
Althoff, T., Mills, D.J., Popot, J.-L., Kühlbrandt, W. (2011) Assembly of electron transport chain components in bovine
mitochondrial supercomplex I 1 III 2 IV 1 . EMBO J. 30:4652–4664.
Anglister, J., Grzesiek, S., Ren, H., Klee, C.B., Bax, A. (1993) Isotope-edited multidimensional NMR of calcineurin B in
the presence of the non-deuterated detergent CHAPS. J. Biomol. NMR 3:121–126.
Arnold, T., Poynor, M., Nussberger, S., Lupas, A.N., Linke, D. (2007) Gene duplication of the eight-stranded β-barrel
OmpX produces a functional pore: a scenario for the evolution of transmembrane β-barrels. J. Mol. Biol.
366:1174–1184.
Arora, A., Abildgaard, F., Bushweller, J.H., Tamm, L.K. (2001) Structure of outer membrane protein A transmembrane
domain by NMR spectroscopy. Nat. Struct. Biol. 8:334–338.
Arunmanee, W., Harris, J.R., Lakey, J.H. (2014) Outer membrane protein F stabilised with minimal amphipol forms
linear arrays and LPS-dependent 2D crystals. J. Membr. Biol. 247:949–956.
Banères, J.-L., Popot, J.-L., Mouillac, B. (2011) New advances in production and functional folding of G protein-coupled
receptors. Trends Biotechnol. 29:314–322.
Bax, A., Kontaxis, G., Tjandra, N. (2001) Dipolar couplings in macromolecular structure determination. Meth. Enzymol.
339:127–174.
490
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins
et al. 2014a), all of which have been validated for attaching MPs onto solid beads or chips, could
conceivably be used to associate MPs to fibers for partial orientation in the magnetic field in view of
residual dipolar coupling measurements (see e.g. Prestegard et al. 2000; Bax et al. 2001; Kay 2001;
Torchia 2015, and refs. therein), or to immobilize them onto resins for the purpose of screening banks
of ligands (cf. Früh et al. 2011).
As mentioned at the beginning of this chapter, no report has yet appeared of applying ssNMR to
APol-trapped MPs. This is to some extent logical: ssNMR is particularly well adapted to studying MPs
in their native membrane environment, and little interest may be found in applying it to MPs that have
been extracted from it. Yet, as has been discussed in Chap. 5, there is some evidence that MPs trapped
in APol in the presence of lipids rebind them, forming ternary complexes in which proteins may exhibit
functional properties closer to those observed in native membranes than in detergent solution
(Martinez et al. 2002; Dahmane et al. 2013). Whereas APols themselves are definitely no membrane
mimics, the interactions experienced by MPs trapped in APols in the presence of lipids may resemble
those experienced in the membrane more than one might think when looking at the chemical structure
of APols. Sensitivity is a major issue in ssNMR. APol-trapped BR can be precipitated without
denaturation either by extensive ultracentrifugation (Gohon et al. 2008) or by deliberately aggregating
the complexes, e.g. by adding Ca
2+ ions to A8-35-trapped BR, or multivalent avidin to BR trapped
with a biotinylated APol (Bazzacco 2009). The concentration of BR in the paste thus obtained is much
higher than can be achieved in membrane samples, a highly favorable factor for ssNMR studies.
19 F
ssNMR has been applied to studying the phosphorylation of an E. coli tyrosine kinase incorporated
into SMALPs (Li et al. 2015).
More generally, one may note that there is a dearth of comparative NMR studies of the same MP
in different environments, on the model of those reported in Etzkorn et al. (2013) (§ 10.3.1.2). A very
interesting suggestion is to use data collected in environments closest to the natural one, such as
ssNMR data collected on membrane-integrated MPs or sNMR data collected on ND-trapped ones, to
assess to which extent other surfactants do or do not affect the structure and dynamics of the MP under
study (Howell et al. 2005; Poget and Girvin 2007; Shenkarev et al. 2010; Etzkorn et al. 2013). Such
comparisons would help delineate which perturbations may or may not be expected from one or the
other surfactant, and help to choose, for each experiment, the type of samples that offers the best
compromise between experimental convenience and reliable information.
References
Althoff, T., Mills, D.J., Popot, J.-L., Kühlbrandt, W. (2011) Assembly of electron transport chain components in bovine
mitochondrial supercomplex I 1 III 2 IV 1 . EMBO J. 30:4652–4664.
Anglister, J., Grzesiek, S., Ren, H., Klee, C.B., Bax, A. (1993) Isotope-edited multidimensional NMR of calcineurin B in
the presence of the non-deuterated detergent CHAPS. J. Biomol. NMR 3:121–126.
Arnold, T., Poynor, M., Nussberger, S., Lupas, A.N., Linke, D. (2007) Gene duplication of the eight-stranded β-barrel
OmpX produces a functional pore: a scenario for the evolution of transmembrane β-barrels. J. Mol. Biol.
366:1174–1184.
Arora, A., Abildgaard, F., Bushweller, J.H., Tamm, L.K. (2001) Structure of outer membrane protein A transmembrane
domain by NMR spectroscopy. Nat. Struct. Biol. 8:334–338.
Arunmanee, W., Harris, J.R., Lakey, J.H. (2014) Outer membrane protein F stabilised with minimal amphipol forms
linear arrays and LPS-dependent 2D crystals. J. Membr. Biol. 247:949–956.
Banères, J.-L., Popot, J.-L., Mouillac, B. (2011) New advances in production and functional folding of G protein-coupled
receptors. Trends Biotechnol. 29:314–322.
Bax, A., Kontaxis, G., Tjandra, N. (2001) Dipolar couplings in macromolecular structure determination. Meth. Enzymol.
339:127–174.
490
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins