observed for both RRS and SERS spectra. The signal enhancement due to SERS is >10
2 Â over that
due to RRS only. It probably reflects the contribution of only the very small fraction of complexes that
find themselves in appropriate positions relative to the silver nanoparticles for SERS to occur and
would be considerably greater if this proportion could be increased (see below, § 8.3).
8.2.3
Fluorescence Spectroscopy
With >20 studies published in >30 publications, fluorescence studies represent, along with UV-visible
absorbance spectroscopy, the bulk of published experiments involving optical spectroscopy
(Table 8.1). Many studies, e.g. Studies 8.1, 8.2, 8.7, and 8.22, rely on fluorescence intensity or
depolarization measurements to determine the K D or kinetics of ligand binding to APol-trapped
receptors or transporters (see Chap. 5, § 5.4). Others, like Studies 8.19, 8.20, or 8.29, have resorted
to fluorescence lifetime or Förster resonance energy transfer (FRET) measurements to examine
conformational changes, oligomerization, and/or aggregation of various APol-trapped MPs. All of
these data have been collected in the presence of unlabeled A8-35, non-ionic APols (NAPols), or
SMALPs (Table 8.1).
Other experiments have relied on the use of APols carrying fluorescent labels, six of which have
been synthesized to date (Table 8.2 and Fig. 8.8).
Fluorescent APols (FAPols) have been used in a wide variety of experiments, among which:
• Examining the interactions of APols with lipid vesicles, using a naphthalene-labeled version
of A8-75 (Vial et al. 2005, 2007, 2009).
• Studying the stability of MP/APol complexes and the exchange of NBD-labeled A8-35
(FAPol NBD ) for detergent or unlabeled APol at the transmembrane (TM) surface of a MP
(Table 8.1, Study 8.5; see Chap. 5, § 5.7).
• Determining the critical aggregation concentration (CAC) of A8-35 as that concentration
above which the formation of particles allows FRET to occur between FAPol NBD and a
rhodamine-labeled version of A8-35 (FAPol rhod ; Study 8.14; see Chap. 4, § 4.3.1.1).
• Using FAPol NBD and a version of A8-35 labeled with Alexa Fluor 647 (FAPol AF647 ) as
tracers in studies of the immobilization of MPs via a version of A8-35 carrying an oligonucleotide tag (OligAPol). Indeed, various versions of A8-35 can be freely mixed, and, by
choosing an appropriate ratio of OligAPol and a FAPol, MP/A8-35 complexes can be
simultaneously tagged and labeled, the OligAPol mediating the immobilization while the
FAPol reports on it (Study 8.23; see Chap. 13, § 13.2.2).
• Probing the topology of a polytopic MP. In Study 8.26, 5-fluorotryptophan (5-F-Trp) residues
were introduced at unique positions in the TM domain of the mannitol permease, EII
mtl (black
and red stars in Fig. 8.9A), in which the four tryptophans present in the wild-type protein, all
located in the transmembrane domain (black circles), had been replaced by phenylalanines.
The transporter was purified and transferred either to unlabeled A8-35 or to fluoresceinlabeled A8-35 (FAPol fluo ). As shown in Fig. 8.9B, C, the extent of quenching of 5-F-Trp
fluorescence by FAPol fluo depended on the position of the residue in the sequence. The data
suggests that position 188 is, on average, closer to the fluorescein groups carried by FAPol fluo
than is position 167, resulting in stronger FRET and quenching. Whereas Study 8.26 was
limited to a proof-of-principle demonstration, systematic investigations of this type could
provide precious topological information about (i) the organization of the TM region and
(ii) transconformations associated with the functional cycle of the protein.
8.2 Optical Spectroscopy Studies of Amphipols and Membrane Protein/Amphipol Complexes
397
2 Â over that
due to RRS only. It probably reflects the contribution of only the very small fraction of complexes that
find themselves in appropriate positions relative to the silver nanoparticles for SERS to occur and
would be considerably greater if this proportion could be increased (see below, § 8.3).
8.2.3
Fluorescence Spectroscopy
With >20 studies published in >30 publications, fluorescence studies represent, along with UV-visible
absorbance spectroscopy, the bulk of published experiments involving optical spectroscopy
(Table 8.1). Many studies, e.g. Studies 8.1, 8.2, 8.7, and 8.22, rely on fluorescence intensity or
depolarization measurements to determine the K D or kinetics of ligand binding to APol-trapped
receptors or transporters (see Chap. 5, § 5.4). Others, like Studies 8.19, 8.20, or 8.29, have resorted
to fluorescence lifetime or Förster resonance energy transfer (FRET) measurements to examine
conformational changes, oligomerization, and/or aggregation of various APol-trapped MPs. All of
these data have been collected in the presence of unlabeled A8-35, non-ionic APols (NAPols), or
SMALPs (Table 8.1).
Other experiments have relied on the use of APols carrying fluorescent labels, six of which have
been synthesized to date (Table 8.2 and Fig. 8.8).
Fluorescent APols (FAPols) have been used in a wide variety of experiments, among which:
• Examining the interactions of APols with lipid vesicles, using a naphthalene-labeled version
of A8-75 (Vial et al. 2005, 2007, 2009).
• Studying the stability of MP/APol complexes and the exchange of NBD-labeled A8-35
(FAPol NBD ) for detergent or unlabeled APol at the transmembrane (TM) surface of a MP
(Table 8.1, Study 8.5; see Chap. 5, § 5.7).
• Determining the critical aggregation concentration (CAC) of A8-35 as that concentration
above which the formation of particles allows FRET to occur between FAPol NBD and a
rhodamine-labeled version of A8-35 (FAPol rhod ; Study 8.14; see Chap. 4, § 4.3.1.1).
• Using FAPol NBD and a version of A8-35 labeled with Alexa Fluor 647 (FAPol AF647 ) as
tracers in studies of the immobilization of MPs via a version of A8-35 carrying an oligonucleotide tag (OligAPol). Indeed, various versions of A8-35 can be freely mixed, and, by
choosing an appropriate ratio of OligAPol and a FAPol, MP/A8-35 complexes can be
simultaneously tagged and labeled, the OligAPol mediating the immobilization while the
FAPol reports on it (Study 8.23; see Chap. 13, § 13.2.2).
• Probing the topology of a polytopic MP. In Study 8.26, 5-fluorotryptophan (5-F-Trp) residues
were introduced at unique positions in the TM domain of the mannitol permease, EII
mtl (black
and red stars in Fig. 8.9A), in which the four tryptophans present in the wild-type protein, all
located in the transmembrane domain (black circles), had been replaced by phenylalanines.
The transporter was purified and transferred either to unlabeled A8-35 or to fluoresceinlabeled A8-35 (FAPol fluo ). As shown in Fig. 8.9B, C, the extent of quenching of 5-F-Trp
fluorescence by FAPol fluo depended on the position of the residue in the sequence. The data
suggests that position 188 is, on average, closer to the fluorescein groups carried by FAPol fluo
than is position 167, resulting in stronger FRET and quenching. Whereas Study 8.26 was
limited to a proof-of-principle demonstration, systematic investigations of this type could
provide precious topological information about (i) the organization of the TM region and
(ii) transconformations associated with the functional cycle of the protein.
8.2 Optical Spectroscopy Studies of Amphipols and Membrane Protein/Amphipol Complexes
397