• The MP(s) that has or have been studied, with their approximate molecular mass. As regards
the latter, no effort has been made to take close account of the contribution of prosthetic
groups, posttranslational modifications, etc., the intent being simply to give the reader an idea
of the size of the proteins that have been handled.
• The type(s) of APols used in the study. As discussed in Chap. 4, poly(styrene-co-maleic acid)
(SMA), alternating diisobutylene/maleic acid copolymer (DIBMA), and NVoy (¼ NV-10)
are listed here under the heading “Amphipols,” as well as amphipathic derivatives of poly-γglutamic acid. Chemical formulae and solution properties of these various polymers are
described in Chap. 4.
• The solution methods that have been used in the study, inasmuch as they fit into the subject
matter of the present chapter. Many studies, for instance, include circular dichroism or
electron microscopy data, which are not necessarily mentioned here (see Chaps. 8 and 12,
respectively). In the “Comments” column, however, some indication is generally given of
what the primary objective of the study was.
The aim of the table is to allow the reader to quickly identify those studies that bear on systems
closest in size or complexity to the one she/he is studying and that have resorted to methods she/he is
considering implementing. Note that the most informative studies tend to be those in which a variety of
techniques have been harnessed to bear on the same system. Indeed, such multidisciplinary studies
provide a good perspective on the limits of and complementarity between various approaches, and they
give precious indications about how to use one to guard against the pitfalls of another. Such are the
cases, for instance, of Study 9.5, with bacteriorhodopsin (BR) as the model system; Study 9.7, on the
cytochrome bc 1 complex; or Study 9.41, on the complexes formed between ExbB and ExbD,
two MPs involved in providing energy from the inner to the outer bacterial membrane to power
nutrient import. BR is a particularly interesting model system, because essentially every polymer that
has ever been developed for use in handling MPs has been tested on it, as well as novel detergents and
other alternative surfactants, e.g. nanodiscs (see Chaps. 2 and 3). It has also been used to test optical
spectroscopy (Chap. 8, § 8.2.2), NMR (Chap. 10, § 10.3.1), and crystallographic (Chap. 11, § 11.3.2)
approaches to studying MPs complexed by APols. The information gathered by the techniques
described in the present chapter has often been key to optimizing the samples to be studied by more
resolving techniques.
9.3
A Walk Through Selected Solution Studies
In order to illustrate the application of the solution methods treated in this chapter to APol-trapped
MPs, I will take as a main guide the first extensive study of this type to have been carried out, that of
BR/A8-35 complexes (Studies 9.1 and 9.5). Key points and alternative approaches will be further
illustrated by data taken from other studies, generally on other MPs. As described in more detail in
Chap. 1, § 1.6.1, BR is a small MP (~27 kDa), folded into a bundle of seven TM α-helices. Retinal, an
aldehyde, is bound covalently but loosely, via a Schiff base, to the ε-amino group of a lysine residue
located in the last TM helix. The Schiff base hydrolyzes spontaneously when the protein denatures,
freeing the retinal, and it reforms, also spontaneously, when the protein refolds and the retinal rebinds.
In the ground state of BR, the Schiff base is protonated, which shifts the absorbance peak of retinal
from ~380 nm, which confers a yellow color to the free cofactor, to the vicinity of 560 nm, making the
holoprotein purple. Upon illumination with visible light, the retinal isomerizes from all-trans to 13-cis,
which initiates a complex cycle of conformational transitions during which the Schiff base proton is
released in the extracellular medium and replaced by a proton taken from the cytosol (see Chap. 1,
410
9 Solution Studies of Membrane Protein/Amphipol Complexes
the latter, no effort has been made to take close account of the contribution of prosthetic
groups, posttranslational modifications, etc., the intent being simply to give the reader an idea
of the size of the proteins that have been handled.
• The type(s) of APols used in the study. As discussed in Chap. 4, poly(styrene-co-maleic acid)
(SMA), alternating diisobutylene/maleic acid copolymer (DIBMA), and NVoy (¼ NV-10)
are listed here under the heading “Amphipols,” as well as amphipathic derivatives of poly-γglutamic acid. Chemical formulae and solution properties of these various polymers are
described in Chap. 4.
• The solution methods that have been used in the study, inasmuch as they fit into the subject
matter of the present chapter. Many studies, for instance, include circular dichroism or
electron microscopy data, which are not necessarily mentioned here (see Chaps. 8 and 12,
respectively). In the “Comments” column, however, some indication is generally given of
what the primary objective of the study was.
The aim of the table is to allow the reader to quickly identify those studies that bear on systems
closest in size or complexity to the one she/he is studying and that have resorted to methods she/he is
considering implementing. Note that the most informative studies tend to be those in which a variety of
techniques have been harnessed to bear on the same system. Indeed, such multidisciplinary studies
provide a good perspective on the limits of and complementarity between various approaches, and they
give precious indications about how to use one to guard against the pitfalls of another. Such are the
cases, for instance, of Study 9.5, with bacteriorhodopsin (BR) as the model system; Study 9.7, on the
cytochrome bc 1 complex; or Study 9.41, on the complexes formed between ExbB and ExbD,
two MPs involved in providing energy from the inner to the outer bacterial membrane to power
nutrient import. BR is a particularly interesting model system, because essentially every polymer that
has ever been developed for use in handling MPs has been tested on it, as well as novel detergents and
other alternative surfactants, e.g. nanodiscs (see Chaps. 2 and 3). It has also been used to test optical
spectroscopy (Chap. 8, § 8.2.2), NMR (Chap. 10, § 10.3.1), and crystallographic (Chap. 11, § 11.3.2)
approaches to studying MPs complexed by APols. The information gathered by the techniques
described in the present chapter has often been key to optimizing the samples to be studied by more
resolving techniques.
9.3
A Walk Through Selected Solution Studies
In order to illustrate the application of the solution methods treated in this chapter to APol-trapped
MPs, I will take as a main guide the first extensive study of this type to have been carried out, that of
BR/A8-35 complexes (Studies 9.1 and 9.5). Key points and alternative approaches will be further
illustrated by data taken from other studies, generally on other MPs. As described in more detail in
Chap. 1, § 1.6.1, BR is a small MP (~27 kDa), folded into a bundle of seven TM α-helices. Retinal, an
aldehyde, is bound covalently but loosely, via a Schiff base, to the ε-amino group of a lysine residue
located in the last TM helix. The Schiff base hydrolyzes spontaneously when the protein denatures,
freeing the retinal, and it reforms, also spontaneously, when the protein refolds and the retinal rebinds.
In the ground state of BR, the Schiff base is protonated, which shifts the absorbance peak of retinal
from ~380 nm, which confers a yellow color to the free cofactor, to the vicinity of 560 nm, making the
holoprotein purple. Upon illumination with visible light, the retinal isomerizes from all-trans to 13-cis,
which initiates a complex cycle of conformational transitions during which the Schiff base proton is
released in the extracellular medium and replaced by a proton taken from the cytosol (see Chap. 1,
410
9 Solution Studies of Membrane Protein/Amphipol Complexes