Amphipol-assisted cell-free synthesis of membrane proteins
(# 2018 by Francis Haraux)
5.3
Composition, Organization, Dynamics, and Solution Properties
of Membrane Protein/Amphipol Complexes
MP/APol complexes have been studied by a vast number of biochemical and biophysical techniques,
including compositional analysis, small-angle neutron and X-ray scattering (respectively SANS and
SAXS), sucrose gradient analytical ultracentrifugation (SG-AUC), sedimentation velocity and equilibrium analytical ultracentrifugation (SV-AUC and Eq-AUC), SEC, DLS, CD, EM after negative
staining (NS-EM) and electron cryomicroscopy (cryo-EM), solution NMR, mass spectrometry (MS),
and many other approaches. Studies that present data on their composition, organization, solution
properties, and dynamics are summarized in Table 5.4.
The most complete data have been collected on BR/A8-35 complexes, with which we will start
(Study 5.16 in Table 5.4). Purple membrane (PM) was solubilized in octylthioglucoside (OTG) and BR
transferred, by adsorbing the detergent onto Bio-Beads, either to plain A8-35 or to isotopically labeled
versions thereof, namely either DAPol, in which the octyl and isopropyl side chains are perdeuterated
(Chap. 4, Fig. 4.12), or [
3 H]A8-35. The native state and functionality of the protein were established by
examining its UV-visible spectrum and photocycle. The particles were characterized by compositional
analysis, SG-, SV-, and Eq-AUC, SANS, and SEC (Gohon et al. 2008). This study was complicated by
the fact that, when it was carried out, not all aspects of the synthesis of A8-35 had been brought under
control. As a result, unknown to the authors, some batches were rendered more hydrophobic than they
ought to have been by the artifactual binding of dicyclohexylurea (DCU), a side product of the grafting
reaction, making some BR/A8-35 complexes, particularly the BR/DAPol ones, prone to aggregation
(see Chap. 4, § 4.2.1, and Protocol 4.1). While seriously complicating the study, this had the useful
consequence of calling attention to the fact that a good monodispersity of free APol particles is a
reliable predictor of the capacity of an APol preparation to form monodisperse MP/APol complexes
(Gohon et al. 2008).
266
5 Formation and Properties of Membrane Protein/Amphipol Complexes
(# 2018 by Francis Haraux)
5.3
Composition, Organization, Dynamics, and Solution Properties
of Membrane Protein/Amphipol Complexes
MP/APol complexes have been studied by a vast number of biochemical and biophysical techniques,
including compositional analysis, small-angle neutron and X-ray scattering (respectively SANS and
SAXS), sucrose gradient analytical ultracentrifugation (SG-AUC), sedimentation velocity and equilibrium analytical ultracentrifugation (SV-AUC and Eq-AUC), SEC, DLS, CD, EM after negative
staining (NS-EM) and electron cryomicroscopy (cryo-EM), solution NMR, mass spectrometry (MS),
and many other approaches. Studies that present data on their composition, organization, solution
properties, and dynamics are summarized in Table 5.4.
The most complete data have been collected on BR/A8-35 complexes, with which we will start
(Study 5.16 in Table 5.4). Purple membrane (PM) was solubilized in octylthioglucoside (OTG) and BR
transferred, by adsorbing the detergent onto Bio-Beads, either to plain A8-35 or to isotopically labeled
versions thereof, namely either DAPol, in which the octyl and isopropyl side chains are perdeuterated
(Chap. 4, Fig. 4.12), or [
3 H]A8-35. The native state and functionality of the protein were established by
examining its UV-visible spectrum and photocycle. The particles were characterized by compositional
analysis, SG-, SV-, and Eq-AUC, SANS, and SEC (Gohon et al. 2008). This study was complicated by
the fact that, when it was carried out, not all aspects of the synthesis of A8-35 had been brought under
control. As a result, unknown to the authors, some batches were rendered more hydrophobic than they
ought to have been by the artifactual binding of dicyclohexylurea (DCU), a side product of the grafting
reaction, making some BR/A8-35 complexes, particularly the BR/DAPol ones, prone to aggregation
(see Chap. 4, § 4.2.1, and Protocol 4.1). While seriously complicating the study, this had the useful
consequence of calling attention to the fact that a good monodispersity of free APol particles is a
reliable predictor of the capacity of an APol preparation to form monodisperse MP/APol complexes
(Gohon et al. 2008).
266
5 Formation and Properties of Membrane Protein/Amphipol Complexes