Step 6 sees the extracted proteins and lipids further diluted with detergent, which is most often
detrimental: MPs become increasingly delipidated, oligomers may come apart, etc. (§ 2.4).
2.3.2
Membrane Protein/Detergent Complexes
The organization of MP/detergent complexes has long remained speculative. It was anticipated that a
belt of detergent would adsorb onto the transmembrane (TM) region of MPs, ensuring the interface
with the aqueous solution and making the proteins water-soluble. This view was supported by many
indirect observations, such as measurements of the amount of bound detergent as a function of the size
of the protein’s TM region or radiation scattering solution studies of MP/detergent complexes
(reviewed in le Maire et al. 2000). This model was directly validated by studying by neutron diffraction
3D crystals of MPs grown from detergent solutions. In X-ray diffraction experiments, detergents
contrast poorly with the surrounding solution: the occasional well-organized molecules of detergent
are hard to identify (see Chap. 1, § 1.5.2), whereas disordered bulk detergent is invisible. Neutron
scattering and diffraction, however, can play on scattering length contrast (see Chap. 9, Box 9.2) to
reveal specific components. At 40% D 2 O, proteins are contrast matched and contribute very little to the
diffraction pattern, whereas the hydrogenated hydrophobic tails of detergents contrast strongly with the
solution. Specific deuteration, when it can be implemented (as regards detergents, see Hiruma-Shimizu
et al. 2015), can be used to enhance or lower the contrast of chosen components or regions. This
approach has been used to determine the distribution of the detergent in MP 3D crystals (Roth et al.
1989, 1991; Pebay-Peyroula et al. 1995; Penel et al. 1998; Prince et al. 2003; Snijder et al. 2003).
As an example, Fig. 2.5B shows an excerpt from a study in which either unlabeled OG or decylN,N
0 -dimethyl amine oxide with a deuterated alkyl chain was used to crystallize trimeric OmpF porin.
Fig. 2.5 Detergent binding to the transmembrane region of trimeric OmpF in three-dimensional crystals.
(A) Atomic structure of the outer membrane trimeric protein OmpF (PDB accession code 2OMF; based on
data in Cowan et al. 1992). Strongly hydrophobic and aromatic amino acid side chains (Leu, Ile, Val, Ala,
Met, Pro, Phe, Tyr, Trp), shown in black, form a ~2.5-nm high belt, which, in situ, faces the hydrophobic
interior of the membrane (horizontal lines) (From Gohon and Popot 2003). (B) The distribution of octyl-βD-glucoside hydrophobic tails around trimeric OmpF, as determined by neutron crystallography (green
cage), fits exactly into the area delimited by the rings of aromatic side chains (orange) at the hydrophobic/
polar boundaries of OmpF’s surface, which probably correspond to the limits of the acyl chains of the lipid
membrane in situ. The porin polypeptide backbone is shown in dark blue (From Pebay-Peyroula et al.
1995, # 1995 Elsevier Ltd. All rights reserved). The composite figure is from Gohon and Popot (2003)
# 2013 Elsevier Ltd. All rights reserved).
68
2 Extracting Membrane Proteins from Their Native Environment
detrimental: MPs become increasingly delipidated, oligomers may come apart, etc. (§ 2.4).
2.3.2
Membrane Protein/Detergent Complexes
The organization of MP/detergent complexes has long remained speculative. It was anticipated that a
belt of detergent would adsorb onto the transmembrane (TM) region of MPs, ensuring the interface
with the aqueous solution and making the proteins water-soluble. This view was supported by many
indirect observations, such as measurements of the amount of bound detergent as a function of the size
of the protein’s TM region or radiation scattering solution studies of MP/detergent complexes
(reviewed in le Maire et al. 2000). This model was directly validated by studying by neutron diffraction
3D crystals of MPs grown from detergent solutions. In X-ray diffraction experiments, detergents
contrast poorly with the surrounding solution: the occasional well-organized molecules of detergent
are hard to identify (see Chap. 1, § 1.5.2), whereas disordered bulk detergent is invisible. Neutron
scattering and diffraction, however, can play on scattering length contrast (see Chap. 9, Box 9.2) to
reveal specific components. At 40% D 2 O, proteins are contrast matched and contribute very little to the
diffraction pattern, whereas the hydrogenated hydrophobic tails of detergents contrast strongly with the
solution. Specific deuteration, when it can be implemented (as regards detergents, see Hiruma-Shimizu
et al. 2015), can be used to enhance or lower the contrast of chosen components or regions. This
approach has been used to determine the distribution of the detergent in MP 3D crystals (Roth et al.
1989, 1991; Pebay-Peyroula et al. 1995; Penel et al. 1998; Prince et al. 2003; Snijder et al. 2003).
As an example, Fig. 2.5B shows an excerpt from a study in which either unlabeled OG or decylN,N
0 -dimethyl amine oxide with a deuterated alkyl chain was used to crystallize trimeric OmpF porin.
Fig. 2.5 Detergent binding to the transmembrane region of trimeric OmpF in three-dimensional crystals.
(A) Atomic structure of the outer membrane trimeric protein OmpF (PDB accession code 2OMF; based on
data in Cowan et al. 1992). Strongly hydrophobic and aromatic amino acid side chains (Leu, Ile, Val, Ala,
Met, Pro, Phe, Tyr, Trp), shown in black, form a ~2.5-nm high belt, which, in situ, faces the hydrophobic
interior of the membrane (horizontal lines) (From Gohon and Popot 2003). (B) The distribution of octyl-βD-glucoside hydrophobic tails around trimeric OmpF, as determined by neutron crystallography (green
cage), fits exactly into the area delimited by the rings of aromatic side chains (orange) at the hydrophobic/
polar boundaries of OmpF’s surface, which probably correspond to the limits of the acyl chains of the lipid
membrane in situ. The porin polypeptide backbone is shown in dark blue (From Pebay-Peyroula et al.
1995, # 1995 Elsevier Ltd. All rights reserved). The composite figure is from Gohon and Popot (2003)
# 2013 Elsevier Ltd. All rights reserved).
68
2 Extracting Membrane Proteins from Their Native Environment