2.5.2
Making Membrane Proteins More Resistant
Making membrane proteins more resistant
(# 2018 by Francis Haraux)
The surfactant is only one of the two partners in MP/detergent complexes. Stability can also be
improved by selecting more resistant proteins. This is in principle beyond the frame of the present
book. However, given that one of the primary interests of resorting to APols is to stabilize the target
MP in aqueous solutions, it is important to know what the alternatives are, as well as to ponder whether
they could be combined.
Working with more stable MPs than one’s primary target, e.g. the human one, is classically
achieved by looking for homologues of the target protein in prokaryotes, whose proteins are usually
less fragile than their eukaryotic counterparts, and, among those, for MPs from thermophilic bacteria,
which evolution has selected for remaining functional under extreme conditions. An alternative
approach is to create and select in the laboratory detergent-resistant mutants of the target protein, a
process that, for reasons to be described below, is known as “conformational thermostabilization”
(Tate 2012). This approach was initiated in the 1990s and early 2000s with such MPs as the TM
segment of the phage M13 coat protein (Deber et al. 1993; Wang and Deber 2000), K
+ channel (Cortes
and Perozo 1997; Perozo et al. 1998), diacylglycerol kinase (DAGK; Lau et al. 1999; Zhou and Bowie
2000), and BR (for a review of these early efforts, see Bowie 2001). It has been applied with
remarkable success to many GPCRs and has led, to date, to the crystallization and structure resolution
of several of them (see for example, Warne et al. 2008; Lebon et al. 2011a, b). The approach looks very
general and could certainly be extended to many other MPs. Before describing it, however, a short
introduction to GPCRs is necessary in order to clarify the nature of the problem to be solved and the
principle of the method. In addition, GPCRs will come up over and over again in subsequent chapters,
and one objective of the present section is to provide a convenient memento to readers who are not
overly familiar with them.
84
2 Extracting Membrane Proteins from Their Native Environment
Making Membrane Proteins More Resistant
Making membrane proteins more resistant
(# 2018 by Francis Haraux)
The surfactant is only one of the two partners in MP/detergent complexes. Stability can also be
improved by selecting more resistant proteins. This is in principle beyond the frame of the present
book. However, given that one of the primary interests of resorting to APols is to stabilize the target
MP in aqueous solutions, it is important to know what the alternatives are, as well as to ponder whether
they could be combined.
Working with more stable MPs than one’s primary target, e.g. the human one, is classically
achieved by looking for homologues of the target protein in prokaryotes, whose proteins are usually
less fragile than their eukaryotic counterparts, and, among those, for MPs from thermophilic bacteria,
which evolution has selected for remaining functional under extreme conditions. An alternative
approach is to create and select in the laboratory detergent-resistant mutants of the target protein, a
process that, for reasons to be described below, is known as “conformational thermostabilization”
(Tate 2012). This approach was initiated in the 1990s and early 2000s with such MPs as the TM
segment of the phage M13 coat protein (Deber et al. 1993; Wang and Deber 2000), K
+ channel (Cortes
and Perozo 1997; Perozo et al. 1998), diacylglycerol kinase (DAGK; Lau et al. 1999; Zhou and Bowie
2000), and BR (for a review of these early efforts, see Bowie 2001). It has been applied with
remarkable success to many GPCRs and has led, to date, to the crystallization and structure resolution
of several of them (see for example, Warne et al. 2008; Lebon et al. 2011a, b). The approach looks very
general and could certainly be extended to many other MPs. Before describing it, however, a short
introduction to GPCRs is necessary in order to clarify the nature of the problem to be solved and the
principle of the method. In addition, GPCRs will come up over and over again in subsequent chapters,
and one objective of the present section is to provide a convenient memento to readers who are not
overly familiar with them.
84
2 Extracting Membrane Proteins from Their Native Environment