3. It is remarkable that crystals can be formed from ternary bc 1 /A8-35/detergent complexes, in
which the surfactant belt carries a high density of surface charges, whereas no crystallographic MP structure has ever been obtained using pure detergents carrying a net charge. On
the one hand, this may suggest that such detergents could possibly be used, if need be, in
combination with neutral or zwitterionic ones. On the other, it bodes well for the use of
non-ionic APols for MP crystallization. Nothing is preventing such studies to be carried out
but for the current absence of large-scale production of these molecules . . . and the will of
funding agencies.
4. Particularly promising are the results achieved upon transferring BR from its complexes with
A8-35 to a mono-olein cubic phase. As tens of MPs have been shown to survive the transfer
from MP/detergent complexes to lipidic mesophases, in which they crystallize, it is reasonable to expect that many MPs purified, folded, or produced in APols will do the same. Given
the high yields observed upon folding MPs in general and GPCRs in particular using APols
(Chap. 6), APol-assisted folding from solubilized inclusion bodies followed by transfer to
lipidic mesophases could well provide a very useful novel route for MP crystallization,
particularly of those MPs that do not stand well being exposed to “non-denaturing”
detergents.
11.4 Protocol: Crystallizing bc 1 /A8-35/Detergent Complexes from Aqueous
Solutions
Given the stabilizing properties of APols, crystallizing MP/APol complexes could give access to the
structure of MPs that cannot be handled in detergents. It has proven itself, however, to be a very
frustrating endeavor. Still, some positive results have been obtained. Whereas attempts at crystallizing
MPs trapped in pure A8-35 have failed, partial success has been achieved with ternary complexes
(§ 11.3.1). We describe here a protocol for the crystallization of cytochrome bc 1 /A8-35/detergent
complexes. The purified bc 1 is first pre-crystallized from detergent solution. Microcrystals are collected and stored. After redissolution, they provide the starting material for crystallization attempts.
The latter are conducted in a mixed APol/detergent environment. In the following, the protocol itself is
printed in roman, whereas comments are in italics and marked with a pointing hand ( ).
11.4.1 Pre-crystallization
At low ionic strength, the bc 1 complex from beef heart mitochondria purified according to Berry
et al. (1991) is only sparingly soluble in detergent solution, especially if the pH is slightly acidic.
Precipitated microcrystals can be collected at high concentration at low ionic strength and buffer
concentration, so that, following redissolution, crystallization conditions will be primarily determined by the nature and concentration of precipitants, surfactants, and additives rather than by
those of the solution the protein was in before precipitation.
Procedure
1. Beef heart mitochondria are purified according to Smith (1967). At the end of bc 1 purification,
conducted according to Berry et al. (1991), run a sizing column to remove aggregates and
11.4 Protocol: Crystallizing bc 1 /A8-35/Detergent Complexes from Aqueous Solutions
523
which the surfactant belt carries a high density of surface charges, whereas no crystallographic MP structure has ever been obtained using pure detergents carrying a net charge. On
the one hand, this may suggest that such detergents could possibly be used, if need be, in
combination with neutral or zwitterionic ones. On the other, it bodes well for the use of
non-ionic APols for MP crystallization. Nothing is preventing such studies to be carried out
but for the current absence of large-scale production of these molecules . . . and the will of
funding agencies.
4. Particularly promising are the results achieved upon transferring BR from its complexes with
A8-35 to a mono-olein cubic phase. As tens of MPs have been shown to survive the transfer
from MP/detergent complexes to lipidic mesophases, in which they crystallize, it is reasonable to expect that many MPs purified, folded, or produced in APols will do the same. Given
the high yields observed upon folding MPs in general and GPCRs in particular using APols
(Chap. 6), APol-assisted folding from solubilized inclusion bodies followed by transfer to
lipidic mesophases could well provide a very useful novel route for MP crystallization,
particularly of those MPs that do not stand well being exposed to “non-denaturing”
detergents.
11.4 Protocol: Crystallizing bc 1 /A8-35/Detergent Complexes from Aqueous
Solutions
Given the stabilizing properties of APols, crystallizing MP/APol complexes could give access to the
structure of MPs that cannot be handled in detergents. It has proven itself, however, to be a very
frustrating endeavor. Still, some positive results have been obtained. Whereas attempts at crystallizing
MPs trapped in pure A8-35 have failed, partial success has been achieved with ternary complexes
(§ 11.3.1). We describe here a protocol for the crystallization of cytochrome bc 1 /A8-35/detergent
complexes. The purified bc 1 is first pre-crystallized from detergent solution. Microcrystals are collected and stored. After redissolution, they provide the starting material for crystallization attempts.
The latter are conducted in a mixed APol/detergent environment. In the following, the protocol itself is
printed in roman, whereas comments are in italics and marked with a pointing hand ( ).
11.4.1 Pre-crystallization
At low ionic strength, the bc 1 complex from beef heart mitochondria purified according to Berry
et al. (1991) is only sparingly soluble in detergent solution, especially if the pH is slightly acidic.
Precipitated microcrystals can be collected at high concentration at low ionic strength and buffer
concentration, so that, following redissolution, crystallization conditions will be primarily determined by the nature and concentration of precipitants, surfactants, and additives rather than by
those of the solution the protein was in before precipitation.
Procedure
1. Beef heart mitochondria are purified according to Smith (1967). At the end of bc 1 purification,
conducted according to Berry et al. (1991), run a sizing column to remove aggregates and
11.4 Protocol: Crystallizing bc 1 /A8-35/Detergent Complexes from Aqueous Solutions
523