without having to measure proton pumping nor time-resolved absorbance changes, is a reliable
indication that the protein is functional.
BR is functional as a monomer. However, when overexpressed in the plasma membrane of
H. salinarum, it associates into two-dimensional crystals formed by the hexagonal arrangement of BR
trimers and lipids, the lipids filling up the center of each trimer and forming a continuous layer between
it and its six neighbors. These 2D crystals, called the “purple membrane,” contain BR as the sole
protein and can be easily purified in large amounts (hundreds of mg of pure BR can be obtained from a
10-L culture). As long as it remains integrated to the purple membrane, BR is remarkably stable.
Denaturation, when it occurs, results in a color change from purple to yellow, due to the spontaneous
hydrolysis of the Schiff base and the release of free retinal in the membrane or detergent environment
(retinal is too hydrophobic to partition significantly in water). This ensemble of properties, combined
with the small size of BR and the relative simplicity of its structure, has made it a favorite material for
the development of biochemical and biophysical approaches to studying the structure and function of
MPs in general. As will be described in the upcoming chapters, BR has proven a very precious test MP
for the development of APols and other nonconventional surfactants.
Fig. 1.24 Overview of the photocycle of bacteriorhodopsin and the proton exchange steps. (A)
Photocycle of BR with the spectral intermediates (K to O), their absorbance maximum (the two M states,
in which the Schiff base is deprotonated, are indistinguishable by absorbance spectroscopy), and their
approximate time scales. The absorbance of a photon by resting-state BR causes the all-trans retinal to
isomerize to a 13-cis configuration, driving a sequence of structural changes within the protein that results
in unidirectional proton transport. (B) 3D structure of BR, with the seven TM helices labeled from A to G,
the location of key residues along the proton-translocation channel, and the accepted sequence of proton
exchange events. Step 1: proton movement from the retinal Schiff base to Asp85. Step 2: proton release to
the extracellular space from the proton release group consisting of Glu194, Glu204, and water molecules.
Step 3: reprotonation of the Schiff base from Asp96. Step 4: proton uptake from the cytosol. Step 5:
deprotonation of Asp85 via proton transfer to the proton release group after the 13-cis retinal has gone back
to its all-trans resting-state configuration (Adapted from Wickstrand et al. 2015).
34
1 Membrane Proteins and Their Natural Environment
indication that the protein is functional.
BR is functional as a monomer. However, when overexpressed in the plasma membrane of
H. salinarum, it associates into two-dimensional crystals formed by the hexagonal arrangement of BR
trimers and lipids, the lipids filling up the center of each trimer and forming a continuous layer between
it and its six neighbors. These 2D crystals, called the “purple membrane,” contain BR as the sole
protein and can be easily purified in large amounts (hundreds of mg of pure BR can be obtained from a
10-L culture). As long as it remains integrated to the purple membrane, BR is remarkably stable.
Denaturation, when it occurs, results in a color change from purple to yellow, due to the spontaneous
hydrolysis of the Schiff base and the release of free retinal in the membrane or detergent environment
(retinal is too hydrophobic to partition significantly in water). This ensemble of properties, combined
with the small size of BR and the relative simplicity of its structure, has made it a favorite material for
the development of biochemical and biophysical approaches to studying the structure and function of
MPs in general. As will be described in the upcoming chapters, BR has proven a very precious test MP
for the development of APols and other nonconventional surfactants.
Fig. 1.24 Overview of the photocycle of bacteriorhodopsin and the proton exchange steps. (A)
Photocycle of BR with the spectral intermediates (K to O), their absorbance maximum (the two M states,
in which the Schiff base is deprotonated, are indistinguishable by absorbance spectroscopy), and their
approximate time scales. The absorbance of a photon by resting-state BR causes the all-trans retinal to
isomerize to a 13-cis configuration, driving a sequence of structural changes within the protein that results
in unidirectional proton transport. (B) 3D structure of BR, with the seven TM helices labeled from A to G,
the location of key residues along the proton-translocation channel, and the accepted sequence of proton
exchange events. Step 1: proton movement from the retinal Schiff base to Asp85. Step 2: proton release to
the extracellular space from the proton release group consisting of Glu194, Glu204, and water molecules.
Step 3: reprotonation of the Schiff base from Asp96. Step 4: proton uptake from the cytosol. Step 5:
deprotonation of Asp85 via proton transfer to the proton release group after the 13-cis retinal has gone back
to its all-trans resting-state configuration (Adapted from Wickstrand et al. 2015).
34
1 Membrane Proteins and Their Natural Environment