Solution Studies of Membrane
Protein/Amphipol Complexes
9
Summary
Prior to using membrane protein/amphipol (MP/APol) complexes for structural or
functional studies, their solution behavior must be characterized. Their mass, size,
shape, composition, internal organization, and interactions can be examined by radiation scattering, ultracentrifugation, size exclusion chromatography, Blue-Native gel
electrophoresis, and other biochemical and biophysical techniques. The implementation of most of these approaches is rather straightforward, as experimental protocols
do not diverge from those used for soluble proteins. Attention should however be paid
to at least two points:
(i) A minimum concentration of free APol is usually necessary to keeping
preparations monodispersed, because total removal of free APol tends to induce
a greater or lesser degree of aggregation.
(ii) With fragile MP complexes, a fine line may have to be walked between conditions,
such as an excess of free APol, that may destabilize complexes and conditions, such
as a deficit of it, that are conducive to aggregation.
Small-angle neutron scattering, a particularly powerful approach, is facilitated,
as compared to studies in detergent solution, by the higher stability of APol-trapped
MPs. It also benefits from the availability of deuterated APols, whose contribution to
scattering can be abolished by matching their scattering length density to that of the
solution while increasing the contrast of the protein with the solvent and reducing the
background noise. However, it is mandatory, in order to collect exploitable data, to
carry out a careful examination of the dispersity of the samples, a task to which
analytical ultracentrifugation is particularly well suited.
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_9
405
Protein/Amphipol Complexes
9
Summary
Prior to using membrane protein/amphipol (MP/APol) complexes for structural or
functional studies, their solution behavior must be characterized. Their mass, size,
shape, composition, internal organization, and interactions can be examined by radiation scattering, ultracentrifugation, size exclusion chromatography, Blue-Native gel
electrophoresis, and other biochemical and biophysical techniques. The implementation of most of these approaches is rather straightforward, as experimental protocols
do not diverge from those used for soluble proteins. Attention should however be paid
to at least two points:
(i) A minimum concentration of free APol is usually necessary to keeping
preparations monodispersed, because total removal of free APol tends to induce
a greater or lesser degree of aggregation.
(ii) With fragile MP complexes, a fine line may have to be walked between conditions,
such as an excess of free APol, that may destabilize complexes and conditions, such
as a deficit of it, that are conducive to aggregation.
Small-angle neutron scattering, a particularly powerful approach, is facilitated,
as compared to studies in detergent solution, by the higher stability of APol-trapped
MPs. It also benefits from the availability of deuterated APols, whose contribution to
scattering can be abolished by matching their scattering length density to that of the
solution while increasing the contrast of the protein with the solvent and reducing the
background noise. However, it is mandatory, in order to collect exploitable data, to
carry out a careful examination of the dispersity of the samples, a task to which
analytical ultracentrifugation is particularly well suited.
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_9
405