Fig. 1.24). As a result, the photocycle of BR creates a TM proton gradient that the bacterium uses as a
source of energy. In solution, the photocycle can be observed by time-resolved absorbance spectroscopy, which provides sensitive measurements of the rate at which each conformational state builds up
and disappears (see e.g. Pocanschi et al. 2006; Dahmane et al. 2013). That the protein is functional can
also be deduced from steady-state spectra, because photocycling results in a slight red shift of the
ground-state absorbance as compared to that observed when the protein has been left to relax in the
dark, due to the conversion of a mixture of states comprising the all-trans and 13-cis retinal isomers to
pure all-trans. The detection of this light-induced bathochromic shift indicates that BR does undergo
its photocycle.
When overproduced in its natural host, the archaebacterium Halobacterium salinarum, BR
accumulates in the plasma membrane in the form of two-dimensional, hexagonal crystals, the
so-called purple membrane (PM) patches, in which BR trimers associate one with another via wellorganized, non-covalently bound lipids. PM patches, which are easily purified and in which BR is
extraordinarily stable, are the most common starting material for biochemical and biophysical studies
of BR. For studies in detergent solutions, BR is solubilized, along with Halobacterium lipids, with a
detergent such as n-octyl-β-D-thioglucopyranoside (OTG).
In the following, examples are given for each of the various techniques that have been applied to
solution studies of MP/APol complexes, going roughly from the most basic to the most informationrich approaches. The principle of each technique is briefly recalled either at the beginning of each
section or in a box. For more in-depth descriptions, the reader is referred to biophysics textbooks,
e.g. Cantor and Schimmel (1980) and Zaccai et al. (2017).
9.3.1
Sucrose Gradient Rate Zonal Ultracentrifugation
The principle of sucrose gradient rate zonal ultracentrifugation, or, more briefly, sucrose gradient
sedimentation velocity (SGSV) measurements, is described in Box 9.1. In Study 9.1, BR in 10 mM
OTG, whose critical micellar concentration (CMC) is ~9 mM, was diluted 2Â with a detergent-free
buffer containing amphipol A8-75, bringing the detergent under its CMC. The preparation was then
layered on a surfactant-free sucrose gradient, containing neither detergent nor APol, and centrifuged
for 10 h. Analysis of the distribution of the protein shows that it migrates as a single band, and none of
it is found in the pellet (Fig. 9.2). The same observation was made with three other MPs solubilized in
various detergents and transferred to A8-75 (Fig. 9.2). In the absence of APol, such experiments result
in complete precipitation of the protein (see Chap. 5, § 5.9.1.5, Fig. 5.42).
Box 9.1 Ultracentrifugation
The principle of ultracentrifugation is to submit a solution of the particles or mixture of particles to
be analyzed to a strong gravity field. There are four main approaches, depending on (i) whether the
particles migrate in a density gradient or a homogeneous column of buffer and (ii) whether they are
spun until an equilibrium distribution is reached or it is the rate at which they migrate that is
analyzed:
• Sucrose gradient sedimentation velocity analysis (SGSV; § 9.3.1, Figs. 9.2, 9.3, 9.4, 9.5,
and 9.6)
A gradient of a dense solute, typically 5–20% w/w sucrose, occasionally glycerol or
iodinated solutes, is formed in centrifuge tubes and the sample deposited on the top. The tubes
9.3 A Walk Through Selected Solution Studies
411
source of energy. In solution, the photocycle can be observed by time-resolved absorbance spectroscopy, which provides sensitive measurements of the rate at which each conformational state builds up
and disappears (see e.g. Pocanschi et al. 2006; Dahmane et al. 2013). That the protein is functional can
also be deduced from steady-state spectra, because photocycling results in a slight red shift of the
ground-state absorbance as compared to that observed when the protein has been left to relax in the
dark, due to the conversion of a mixture of states comprising the all-trans and 13-cis retinal isomers to
pure all-trans. The detection of this light-induced bathochromic shift indicates that BR does undergo
its photocycle.
When overproduced in its natural host, the archaebacterium Halobacterium salinarum, BR
accumulates in the plasma membrane in the form of two-dimensional, hexagonal crystals, the
so-called purple membrane (PM) patches, in which BR trimers associate one with another via wellorganized, non-covalently bound lipids. PM patches, which are easily purified and in which BR is
extraordinarily stable, are the most common starting material for biochemical and biophysical studies
of BR. For studies in detergent solutions, BR is solubilized, along with Halobacterium lipids, with a
detergent such as n-octyl-β-D-thioglucopyranoside (OTG).
In the following, examples are given for each of the various techniques that have been applied to
solution studies of MP/APol complexes, going roughly from the most basic to the most informationrich approaches. The principle of each technique is briefly recalled either at the beginning of each
section or in a box. For more in-depth descriptions, the reader is referred to biophysics textbooks,
e.g. Cantor and Schimmel (1980) and Zaccai et al. (2017).
9.3.1
Sucrose Gradient Rate Zonal Ultracentrifugation
The principle of sucrose gradient rate zonal ultracentrifugation, or, more briefly, sucrose gradient
sedimentation velocity (SGSV) measurements, is described in Box 9.1. In Study 9.1, BR in 10 mM
OTG, whose critical micellar concentration (CMC) is ~9 mM, was diluted 2Â with a detergent-free
buffer containing amphipol A8-75, bringing the detergent under its CMC. The preparation was then
layered on a surfactant-free sucrose gradient, containing neither detergent nor APol, and centrifuged
for 10 h. Analysis of the distribution of the protein shows that it migrates as a single band, and none of
it is found in the pellet (Fig. 9.2). The same observation was made with three other MPs solubilized in
various detergents and transferred to A8-75 (Fig. 9.2). In the absence of APol, such experiments result
in complete precipitation of the protein (see Chap. 5, § 5.9.1.5, Fig. 5.42).
Box 9.1 Ultracentrifugation
The principle of ultracentrifugation is to submit a solution of the particles or mixture of particles to
be analyzed to a strong gravity field. There are four main approaches, depending on (i) whether the
particles migrate in a density gradient or a homogeneous column of buffer and (ii) whether they are
spun until an equilibrium distribution is reached or it is the rate at which they migrate that is
analyzed:
• Sucrose gradient sedimentation velocity analysis (SGSV; § 9.3.1, Figs. 9.2, 9.3, 9.4, 9.5,
and 9.6)
A gradient of a dense solute, typically 5–20% w/w sucrose, occasionally glycerol or
iodinated solutes, is formed in centrifuge tubes and the sample deposited on the top. The tubes
9.3 A Walk Through Selected Solution Studies
411