complexes, and the formation of homogeneous MP/ND complexes is far from trivial (see e.g. Raschle
et al. 2010; Warschawski et al. 2011; Etzkorn et al. 2013; Hagn et al. 2013; Catoire et al. 2014; Viegas
et al. 2016). Variants of scaffold proteins that form smaller or more stable NDs have been developed
(see Chap. 3, § 3.3.2). A succinct overview of NMR studies of MPs trapped in NDs is presented in
Chap. 3 (§ 3.3.5). Several NMR laboratories have introduced the interesting suggestion that spectra
collected on MPs trapped either in bicelles (Howell et al. 2005; Poget and Girvin 2007) or in NDs
(Shenkarev et al. 2010), which are more complicated to implement and do not necessarily yield the best
spectra, but which provide MPs with a more membrane-like environment, be used as a benchmark to
evaluate, by comparing 2D
1 H,
15 N-TROSY spectra, the ability of detergents to maintain the native
structure of target MPs for which there is no easy in vitro functional assay.
Organic solvents offer the potential advantage of a low viscosity, which can considerably
accelerate tumbling, and the drawback of being poor substitutes for the membrane environment.
They have been used occasionally, according to either of two configurations. In the first one, the
MP is directly solubilized in a solvent like trifluoroethanol (TFE), at the risk of affecting its structure
(see e.g. Shenkarev et al. 2010). In the second one, each MP binds two reverse micelles (Fig. 10.2C).
The water phase inside the micelles interacts with the extramembrane regions of the MP, the organic
solvent with the TM one. There is currently too little experience with this system to evaluate the risk of
MP structure modification, but the latter should not be underestimated, as organic solvents can
interfere with protein/protein and protein/lipid interactions (see e.g. Kang and Li 2011; Qureshi and
Goto 2011, and refs. therein).
Within each of these systems, optimizing the choice of the surfactant and experimental
conditions is an essential and difficult task (see e.g. Poget and Girvin 2007; Nietlispach and Gautier
2011; Qureshi and Goto 2011). This is illustrated in Figs. 10.3 and 10.4 by two examples taken from
the work of Daniel Nietlispach and Antoine Gautier on sensory rhodopsin II (pSRII), a MP with seven
TM α-helices and a mass of 26.7 kDa (Gautier et al. 2010; Nietlispach and Gautier 2011). Figure 10.3
illustrates two effects, that of the choice of the detergent (compare the first four panels with the last
two) and that of the temperature: the last two spectra were obtained in the same detergent, diC 7 PC, but
spectrum E was collected at 40
C, spectrum F at 50
C.
Figure 10.4 illustrates, on a given system – sensory rhodopsin in diC 7 PC, the effect of varying
the detergent concentration. As the concentration is increased from 16.5 mM (33Â the CMC of
diC 7 PC) to 55 mM (110Â the CMC), the resolution of the resonance peaks keeps increasing. At
82.5 mM (165Â the CMC), it decreases again, because of the increasing viscosity of the detergent
solution. These two series of experiments illustrate one of the difficulties associated with studying MPs
by sNMR in detergent solutions: conditions that improve the resolution of the spectra, such as
increasing the detergent concentration and raising the temperature, are also conditions which, as we
have seen in Chap. 2, accelerate MP inactivation.
Despite these difficulties, rapid progress is being made, helped by the development of higher field
NMR magnets, cryogenic probes, improved pulse sequences, and labeling schemes, so that solving de
novo the structure of a 30–40 kDa MP is no longer out of reach (see e.g. Kim et al. 2009; Kang and Li
2011; Nietlispach and Gautier 2011; Qureshi and Goto 2011; Warschawski et al. 2011); for a recent
review of MP structures studied by ssNMR, see Patching 2015; for up-to-date lists of NMR structures of
MPs, see the sites maintained by Dror E. Warschawski (http://www.drolist.com/nmr/MPNMR.html)
and by Stephen H. White (http://blanco.biomol.uci.edu/mpstruc/). A most interesting survey of
relatively recent sNMR studies of α-helical MPs is given in Nietlispach and Gautier 2011 (see also
Kim et al. 2009; Qureshi and Goto 2011; Warschawski et al. 2011). Out of the seven MPs listed for
which complete assignments were obtained, three are monomeric, the heaviest one being pSRII,
458
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins
et al. 2010; Warschawski et al. 2011; Etzkorn et al. 2013; Hagn et al. 2013; Catoire et al. 2014; Viegas
et al. 2016). Variants of scaffold proteins that form smaller or more stable NDs have been developed
(see Chap. 3, § 3.3.2). A succinct overview of NMR studies of MPs trapped in NDs is presented in
Chap. 3 (§ 3.3.5). Several NMR laboratories have introduced the interesting suggestion that spectra
collected on MPs trapped either in bicelles (Howell et al. 2005; Poget and Girvin 2007) or in NDs
(Shenkarev et al. 2010), which are more complicated to implement and do not necessarily yield the best
spectra, but which provide MPs with a more membrane-like environment, be used as a benchmark to
evaluate, by comparing 2D
1 H,
15 N-TROSY spectra, the ability of detergents to maintain the native
structure of target MPs for which there is no easy in vitro functional assay.
Organic solvents offer the potential advantage of a low viscosity, which can considerably
accelerate tumbling, and the drawback of being poor substitutes for the membrane environment.
They have been used occasionally, according to either of two configurations. In the first one, the
MP is directly solubilized in a solvent like trifluoroethanol (TFE), at the risk of affecting its structure
(see e.g. Shenkarev et al. 2010). In the second one, each MP binds two reverse micelles (Fig. 10.2C).
The water phase inside the micelles interacts with the extramembrane regions of the MP, the organic
solvent with the TM one. There is currently too little experience with this system to evaluate the risk of
MP structure modification, but the latter should not be underestimated, as organic solvents can
interfere with protein/protein and protein/lipid interactions (see e.g. Kang and Li 2011; Qureshi and
Goto 2011, and refs. therein).
Within each of these systems, optimizing the choice of the surfactant and experimental
conditions is an essential and difficult task (see e.g. Poget and Girvin 2007; Nietlispach and Gautier
2011; Qureshi and Goto 2011). This is illustrated in Figs. 10.3 and 10.4 by two examples taken from
the work of Daniel Nietlispach and Antoine Gautier on sensory rhodopsin II (pSRII), a MP with seven
TM α-helices and a mass of 26.7 kDa (Gautier et al. 2010; Nietlispach and Gautier 2011). Figure 10.3
illustrates two effects, that of the choice of the detergent (compare the first four panels with the last
two) and that of the temperature: the last two spectra were obtained in the same detergent, diC 7 PC, but
spectrum E was collected at 40
C, spectrum F at 50
C.
Figure 10.4 illustrates, on a given system – sensory rhodopsin in diC 7 PC, the effect of varying
the detergent concentration. As the concentration is increased from 16.5 mM (33Â the CMC of
diC 7 PC) to 55 mM (110Â the CMC), the resolution of the resonance peaks keeps increasing. At
82.5 mM (165Â the CMC), it decreases again, because of the increasing viscosity of the detergent
solution. These two series of experiments illustrate one of the difficulties associated with studying MPs
by sNMR in detergent solutions: conditions that improve the resolution of the spectra, such as
increasing the detergent concentration and raising the temperature, are also conditions which, as we
have seen in Chap. 2, accelerate MP inactivation.
Despite these difficulties, rapid progress is being made, helped by the development of higher field
NMR magnets, cryogenic probes, improved pulse sequences, and labeling schemes, so that solving de
novo the structure of a 30–40 kDa MP is no longer out of reach (see e.g. Kim et al. 2009; Kang and Li
2011; Nietlispach and Gautier 2011; Qureshi and Goto 2011; Warschawski et al. 2011); for a recent
review of MP structures studied by ssNMR, see Patching 2015; for up-to-date lists of NMR structures of
MPs, see the sites maintained by Dror E. Warschawski (http://www.drolist.com/nmr/MPNMR.html)
and by Stephen H. White (http://blanco.biomol.uci.edu/mpstruc/). A most interesting survey of
relatively recent sNMR studies of α-helical MPs is given in Nietlispach and Gautier 2011 (see also
Kim et al. 2009; Qureshi and Goto 2011; Warschawski et al. 2011). Out of the seven MPs listed for
which complete assignments were obtained, three are monomeric, the heaviest one being pSRII,
458
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins