Extracting Membrane Proteins from Their
Native Environment
2
Summary
Whereas many functional studies and some structural ones can be carried out while
keeping membrane proteins in their native environment, other investigations require
them to be extracted from the membrane, purified, and handled in aqueous solutions.
The usual route is to resort to detergents. Detergents are small amphiphiles that, in
aqueous solutions, associate into particles, called micelles, into which lipids and
membrane proteins can be solubilized. A major difficulty in the use of detergents is
that they must be “strong” enough to disrupt membranes and disperse their
components and “mild” enough not to inactivate the target proteins, a balance that
is often quite difficult to achieve. In this chapter, we will review the mechanisms by
which detergents extract membrane proteins, the nature of the complexes they form
with them, the reasons why detergent-solubilized membrane proteins are often shortlived, and the measures that can be taken to confer them enough stability for experimental investigations to be carried out.
2.1
Introduction
Many studies of membrane proteins (MPs) can be carried out in their cellular environment or on native
membrane fragments. In the first case, the protein is exposed to the same or nearly the same aqueous
and membrane environments as in the whole organism. In the second, a number of important
interactions may have been lost, such as exposure to gradients of concentration of molecules and
ions, of pH, or of redox potential, to the transmembrane electric field, to interactions with cytosolic or
extracellular molecules, etc. These factors may affect the protein’s function and, sometimes, its
structure. Upon breaking cells open, MP cytosolic and extramembrane domains, for instance, become
exposed to the same redox potential; in vivo, the former are exposed to a reducing medium and may
comprise free sulfhydryls, whereas the latter, on the contrary, are exposed to an oxidizing environment
and often feature disulfide bridges. Artifactual bridges between cytosolic cysteine residues may form
upon breaking the cells open in an air-saturated buffer, whereas extracellular bridges may be reduced
by the reductants freed from the cytosol. This can be prevented by homogenizing the tissue in the
presence of iodoacetamide but at the cost of blocking free thiols that may be functionally important.
# 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_2
59
Native Environment
2
Summary
Whereas many functional studies and some structural ones can be carried out while
keeping membrane proteins in their native environment, other investigations require
them to be extracted from the membrane, purified, and handled in aqueous solutions.
The usual route is to resort to detergents. Detergents are small amphiphiles that, in
aqueous solutions, associate into particles, called micelles, into which lipids and
membrane proteins can be solubilized. A major difficulty in the use of detergents is
that they must be “strong” enough to disrupt membranes and disperse their
components and “mild” enough not to inactivate the target proteins, a balance that
is often quite difficult to achieve. In this chapter, we will review the mechanisms by
which detergents extract membrane proteins, the nature of the complexes they form
with them, the reasons why detergent-solubilized membrane proteins are often shortlived, and the measures that can be taken to confer them enough stability for experimental investigations to be carried out.
2.1
Introduction
Many studies of membrane proteins (MPs) can be carried out in their cellular environment or on native
membrane fragments. In the first case, the protein is exposed to the same or nearly the same aqueous
and membrane environments as in the whole organism. In the second, a number of important
interactions may have been lost, such as exposure to gradients of concentration of molecules and
ions, of pH, or of redox potential, to the transmembrane electric field, to interactions with cytosolic or
extracellular molecules, etc. These factors may affect the protein’s function and, sometimes, its
structure. Upon breaking cells open, MP cytosolic and extramembrane domains, for instance, become
exposed to the same redox potential; in vivo, the former are exposed to a reducing medium and may
comprise free sulfhydryls, whereas the latter, on the contrary, are exposed to an oxidizing environment
and often feature disulfide bridges. Artifactual bridges between cytosolic cysteine residues may form
upon breaking the cells open in an air-saturated buffer, whereas extracellular bridges may be reduced
by the reductants freed from the cytosol. This can be prevented by homogenizing the tissue in the
presence of iodoacetamide but at the cost of blocking free thiols that may be functionally important.
# 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_2
59