Archives of Biochemistry and Biophysics 564 (2014) 314–326
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Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Review
Folding membrane proteins in vitro: A table and some comments
Jean-Luc Popot
Centre National de la Recherche Scientifique/Université Paris-7 UMR 7099, Institut de Biologie Physico-Chimique (FRC 550), 13, rue Pierre-et-Marie-Curie, F-75005 Paris, France
a r t i c l e
i n f o
Article history:
Received 25 April 2014
and in revised form 17 June 2014
Available online 2 July 2014
Keywords:
Membrane proteins
Surfactants
Folding
Biochemistry
Biophysics
a b s t r a c t
Thirty-three years have elapsed since the first membrane protein (MP) was brought back in vitro to its
native state starting from the completely unfolded polypeptide. Folding MPs is as useful from a practical
point of view as it is thought-provoking from a theoretical one. Yet, this activity is considered as a high-risk,
time-consuming endeavor, full of pitfalls, its path littered with the broken careers of graduate students sacrificed on the altar of a long shot that never paid off. In fact, a surprisingly high number of MPs have actually
been folded or refolded in vitro. Analysis of the literature indicates (i) that the endeavor is not as desperate as
it may seem, (ii) that techniques are diversifying and improving, and (iii) that many MPs do not need the
cellular biosynthetic apparatus, nor even a membrane environment, to reach a functional 3D structure. A
compilation, hopefully close to complete, is presented of MPs that have been (re)folded in vitro to-date, with
the conditions of their synthesis, the denaturant(s) used, if any, and the (re)folding conditions, along with a
few comments. The hope is that this analysis will encourage membrane protein biochemists to consider
producing their target proteins in this way, help them decide about an experimental course, and stimulate
the reflection about which environments favor membrane protein folding and why.
Ó 2014 Elsevier Inc. All rights reserved.
Aim of the review
While working on a couple of papers dealing with the use of
amphipols to fold membrane proteins (MPs),1 I felt the need to
patch my rather holey knowledge of what had been going on in this
field using other folding media. I was primarily interested in two
points: (i) Which types of MPs have been folded in vitro? (ii) Which
media have proven adequate to fold at least one MP out of a cell? I
naively thought that I could catch up on the literature by going
through a few reviews. I discovered that, whereas there is no dearth
of reviews on MP folding (among relatively recent ones, see e.g. Refs.
[1–11]), none of them has elected to provide a complete survey of
the two points I was interested in. Closest to it are two excellent
reviews devoted to b-barrel MPs [12,13]. Both of them have been a
precious resource for the present compilation, but they do not cover
the entire field. My second move was as naive as the first. I was
under the mistaken impression, from the papers I had come across
and the presentations I had heard, that the total number of MPs that
E-mail address: [email protected]
Abbreviations used: MP, membrane protein; CFE, cell-free expression; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; SDS–PAGE, SDS–
polyacrylamide gel electrophoresis; SDS, sodium dodecylsulfate; GdnHCl, guanidine
hydrochloride; IBs, inclusion bodies; LDS, lithium dodecylsulfate; BR, bacteriorhodopsin; 2D, two-dimensional; CD, circular dichroism; LHCII, light-harvesting complex
II; OmpF, outer membrane protein F; APols, amphipols, NDs, nanodiscs; TFE,
trifluoroethanol; SMFS, single-molecule force spectroscopy.
1
http://dx.doi.org/10.1016/j.abb.2014.06.029
0003-9861/Ó 2014 Elsevier Inc. All rights reserved.
had been proven to be foldable in vitro did not exceed 20–40. It
should not take that long to hunt down all of them and compile
my catch in a little table, which would permit me to clarify my views
about what has been made to work and what not, and, if published,
could be useful to colleagues interested in this field. As will appear
below, the little table turned out to be quite long, comprising, as
of today, nearly 90 significantly different MPs, and a good dozen
unfolding and folding procedures. The result far exceeded the frame
of the short discussion paper I had initially in mind. Having been
kindly invited by Daniel Otzen to contribute to the present special
issue of Arch. Biochem. Biophys. on Folding and stability of integral
membrane proteins, I proposed to seize this opportunity to put the
results of this literature search at the disposal of my colleagues.
The aim of this survey is therefore to compile, as completely as
possible, a list of all MPs that have been brought to a folded, presumably native state, while indicating the state each of them
was in before folding, the medium it was folded in, along with
some information about the folding procedure used. Asserting to
which extent a MP has been renatured or folded can be difficult,
particularly when it has no enzymatic activity. I have retained only
cases where sufficiently stringent functional or structural tests
have been applied, such as ligand binding or oligomerization,
X-ray or NMR data, etc. The yields however can be difficult to
determine and are often not specified in the original papers. They
have not been included in the table. Cell-free expression (CFE) data
can be particularly frustrating, because tens of MPs are often
J.-L. Popot / Archives of Biochemistry and Biophysics 564 (2014) 314–326
tested, but data can be limited to visualizing bands following
SDS–polyacrylamide gel electrophoresis (SDS–PAGE), and ligandbinding or other functional tests, when they have been carried
out, are seldom quantitative. I may well have erred one way or
another when attempting to select particularly convincing cases.
It is not a matter of great concern, I think, because what we are
interested in here is a survey of what has been made to work
and what not, and the fact that the number of G protein-coupled
receptor that have been effectively expressed in vitro and shown
to bind specific ligands may have been somewhat over- or underestimated, for instance, is not the essential point. Readers desirous
of more details about CFE results are referred to reviews by specialists (for relatively recent ones, see e.g. Refs. [14–21a]).
In the following, I will keep, whenever appropriate, to a distinction between ‘‘folding’’ and ‘‘refolding’’. ‘‘Refolding’’ applies to
experiments in which a MP has been first obtained in its native
form and then denatured using such chemicals as sodium dodecylsulfate (SDS), guanidine hydrochloride (GdnHCl) or urea, before
being brought back to a native-like state. ‘‘Folding’’ applies to
MPs that had never achieved their native state before in vitro
folding, as is the case for MPs folded from inclusion bodies (IBs)
or produced by CFE.
Denaturants
Whereas many treatments can induce MP denaturation,
relatively few of them yield preparations that are amenable to
refolding. Heat, for instance, is classically used to compare the stability in a given environment of various MPs or MP mutants, or that
of the same protein in different media or in the presence of various
ligands. However, heat denaturation of proteins results in various
modifications, including chemical modifications and the formation
of aggregates, which are generally irreversible (see e.g. Refs.
[21b–e], and references therein). Organic solvents can be used to
denature MPs, but refolding is almost never performed directly
from them (see below).
What is meant by a ‘‘denatured’’ state can be ambiguous. Very
little provocation is needed to shift a MP from a functional state
to a non-functional one, and this does not necessarily involve large
changes of conformation. In principle, the claim that a MP can be
folded in vitro should include the demonstration that the denatured state was really fully unfolded, and could not have retained
any structural information beyond the primary structure. It is useful therefore to say a few words of what happens when a protein is
exposed to either of the three most classically used denaturants,
SDS, GdnHCl, and urea.
SDS – and, when one wants to work at relatively high concentrations in the cold, lithium dodecylsulfate (LDS), which does not
precipitate at 4 °C – binds to polypeptide chains, which, as a result,
lose most of their tertiary structure. A random-coil state is not
achieved, however, because association of a polypeptide with
dodecylsulfate tends to favor the formation of a-helices [22]. Typically, a primarily a-helical MP will lose helicity when exposed to
SDS (see e.g. Ref. [23]), whereas a primarily b-stranded MP will
acquire some [24]. Studies with non-membrane proteins suggest
that protein/SDS complexes form chains of beads, each bead comprising a peptide segment associated to the detergent (see e.g. Refs.
[25,26a], and references therein). The fact that most soluble globular proteins migrate, during SDS–PAGE, at a speed that inversely
correlates with the length of the polypeptide suggests that they
all form with SDS the same type of complexes, in which all or most
tertiary interactions are lost. It is a common observation, however,
that polytopic a-helical MPs tend to migrate, upon SDS–PAGE, differently from what their mass would lead one to expect (generally
faster; sometimes slower; for discussions about the origin(s) of this
315
effect, see e.g. Refs. [26b,26c]). This may be taken to suggest that
they unfold less extensively than soluble proteins, and, by inference, that their transmembrane helices are more resistant to SDS.
It is not known to which extent the a-helical segments present
in an SDS-denatured a-helical MP overlap with a-helices that were
present in the native protein, but a significant degree of overlap is
suggested by the fact that peptides corresponding to bacteriorhodopsin (BR) transmembrane helices have been shown by NMR to
adopt an a-helical structure in SDS [27,28]. It is a frequent observation that heating a polytopic a-helical MP in SDS and bringing it
back to room temperature before running an SDS–PAGE generates
multimers. This indicates that SDS is unable, at room temperature,
to prevent the formation of some intermolecular interactions and
suggests that certain tertiary interactions, presumably between
hydrophobic helices, may well survive SDS denaturation. Some
MPs, in particular many b-barrel ones, resist unfolding by SDS at
room temperature. This is very useful experimentally, because
the simple device of comparing the protein’s migration upon
SDS–PAGE with or without heating the sample provides information about whether it was folded vs. unfolded, or monomeric vs.
oligomeric, before being heated.
The situation seems simpler as regards urea and GdnHCl, both
of which, at high concentrations (typically 8 M urea or 4–6 M
GdnHCl), induce, in soluble proteins, the formation of random coils
[29]. However, the unfolding of transmembrane b-barrels is not
necessarily complete [30]. Unfolding of a-helical MPs by urea is
very incomplete [31,32a], GdnCl (8 M) seeming more efficient [31].
In the beginning was Gobind Khorana
In vitro folding of an integral MP from a completely unfolded
state was first described in 1981 by the laboratory of H.G. Khorana,
at the MIT. Khorana had shared the Nobel prize in 1968 with
Robert W. Holley and Marshall W. Nirenberg for deciphering the
genetic code. He then turned to trying to understand the molecular
mechanism of vision, which brought him to develop extensively
the chemistry and biochemistry of MPs. It is useful to replace this
first folding demonstration in the context of the time. In the late
seventies, not only was no atomic 3D structure of any MP known,
but only one primary structure had been established, that of glycophorin A, a dimeric MP whose monomer comprises a single
transmembrane a-helix. The biochemistry of isolating MPs in a
functional state was still in its infancy, relying largely on the pioneering but largely empirical recipes of the Racker era [32b].
Why so many MPs lost functionality almost as soon as extracted
from membranes was not understood.
In the early seventies, BR, a marvelous model MP, was discovered and studied in Walter Stoeckenius’ laboratory at the UCSF
(reviewed in Refs. [33,34]). BR, a polytopic MP, accumulates in
the plasma membrane of the archaebacterium Halobacterium salinarum (then Halobacterium halobium), where, if overproduced, it
assembles into highly ordered two-dimensional (2D) crystals, the
so-called ‘‘purple membrane’’. BR possesses the same cofactor as
vertebrate rhodopsin, retinal. Retinal is bound to the protein by a
protonated Schiff base, which shifts its absorption spectrum by
almost 200 nm to the red, turning its color from yellow to purple.
Upon denaturing BR, free retinal is released from the unfolded apoprotein (bacterio-opsin; BO) and the sample turns from purple to
yellow. Upon renaturation, retinal spontaneously rebinds to
refolded BO, and the sample turns back from yellow to purple,
which greatly facilitates renaturation studies. Furthermore, BR
can easily be produced in large amounts – hundreds of mg –, and
it is very stable when in the purple membrane, making it a remarkably convenient experimental material for biochemists, biophysicists, and structural biologists (a search of PubMed with the sole
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keyword ‘‘bacteriorhodopsin’’ turns up close to 4000 references).
The function of BR – establishing a transmembrane proton gradient
using the energy of light – was soon established by Dieter Oesterhelt & Walter Stoeckenius. To which extent BR is a good model for
rhodopsin was long debated. Despite an overall structural similarity – same number of transmembrane a-helices – and the presence
of the same cofactor bound, in both cases, to the most C-terminal
a-helix, their functions are not the same, they present no sequence
homology, and their tertiary structures are significantly different.
Nonetheless, BR has been and still is extensively used as a remarkable model for establishing experimental methods and principles
that can then be tentatively extended to other MPs, including in
the field of folding.
In the mid-seventies, Richard Henderson and Nigel Unwin
established, by electron microscopy, a low-resolution 3D map of
BR showing that it features seven transmembrane a-helices.
Strange as it may seem today, this 3D structural information was
obtained several years before the primary structure of BR was
known. In the seventies, sequencing by molecular genetics did
not exist. Protein sequences had to be established the hard way,
by cutting the polypeptide into pieces, sequencing chemically the
resulting peptides, and putting the whole puzzle together. For
MPs, this was a formidable task, because of the insolubility of the
transmembrane fragments. Gobind Khorana, a chemist by training,
endeavored to establish the amino acid sequence of BR, in hot competition with the Russian Yuri Ovchinnikov. After years of very
hard work, the two groups succeeded in 1979. In the process, they
had identified ways to handle denatured BR and its fragments in
organic solvents or in SDS solutions, which provided the platform
from which refolding experiments could be launched.
At this point, it is useful to backtrack by a couple of decades and
to remind one of what had been going on as regards the folding of
soluble proteins. Taking ribonuclease as a model, Christian Anfinsen and his colleagues, in the sixties, had shown that this enzyme,
after its eight disulfide bridges had been reduced and the polypeptide unfolded in urea, could recover full activity in vitro when the
denaturant was removed and the bridges allowed to reoxidize
[35–37]. This established that the native structure of the enzyme
is not dictated by the biosynthetic apparatus, but by the interaction
of the amino acid sequence with itself and with its environment,
and that it corresponds to the (or a) free energy minimum of this
ensemble. It was totally uncertain, however, whether the same rule
could apply to MPs, whose synthesis and membrane insertion take
place in a highly anisotropic medium and are catalyzed by a complex apparatus whose composition and role just started to be
unraveled in the seventies. That MPs could be kinetically blocked,
for instance because some of their regions cannot flip through the
membrane, in a native conformation that does not correspond to
the free-energy minimum was a real possibility. In such a case,
refolding in vitro would have been extremely complex, if not
impossible. The work carried out at the MIT in the early 80s
showed that it was not.
In their epoch-making 1981 paper [23], Khorana and colleagues
showed that SDS-denatured BR could be brought back to a retinal-binding state following dilution of the SDS into a large excess of a mixture of
‘‘non-denaturing’’ detergent (initially bile salts, then 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) and lipids
(initially soybean lipids, then dimyristoylphosphatidylcholine or purple
membrane lipids) [23,38,39]. Following removal of the detergents and
formation of vesicles, light-induced proton-pumping could be demonstrated. This could pass as half-a-demonstration, however,
because BR retains in SDS about 2/3 of its initial a-helical content.
It could therefore be argued that an SDS-unfolded a-helical MP
has retained some memory of its folded state, which could serve
as a starting point for refolding upon removal of SDS. This objection
was put to rest in Huang’s paper by showing that BR could be fully
unfolded in organic solvent – as shown by circular dichroism (CD)
and NMR –, then transferred to SDS, where it recovered some
a-helical structure, and then fully refolded by diluting the SDS into
lipid/cholate mixed micelles. SDS, in such a succession of steps, can
be seen as providing an intermediate, partially prefolded stage,
from which the folded protein can form [23]. Huang et al. also
described, in the same article, how active BR could be refolded from
two fragments, obtained by chymotryptic cleavage between the
second and the third transmembrane helices, an observation that
was to have a rich posterity. It is remarkable, when reading this
work in a period plagued with the tyranny of short formats and high
impact factors, that this memorable piece of work was not
machete-mangled to fit into the procrustean bed of a top-visibility
journal: it was submitted to J. Biol. Chem. as a full-length, well-documented article, with enough background to put it into perspective
and the necessary experimental details for later experimenters to
be able to reproduce it and build on it.
Six years would elapse before the next a-helical MP was
refolded, namely light-harvesting complex II (LHCII) from spinach
chloroplasts [40]. It was, again, a feat, LHCII being a trimer carrying
numerous cofactors. The procedure used was rather complex and
has not been frequently employed afterwards, involving freeze–
thaw cycles of a mixture of protein, lipids and cofactors in the
presence of LDS. It was used again to fold pea LHCII obtained as
inclusion bodies [41], but was replaced later by a relatively more
mainstream approach, precipitation of dodecylsulfate in the
presence of detergent micelles [42].
What about b-barrel membrane proteins?
Somewhat amazingly, one of the first two b-barrel MPs to be
refolded, in 1990, was an oligomeric MP, outer membrane protein F (OmpF) from Escherichia coli, a trimeric porin [24]. OmpF,
which is nearly fully b-stranded, was denatured either in GdnHCl,
where it forms random coils, or in SDS, where it acquires some
a-helicity, and renatured by transfer to lipid/detergent mixed
micelles. In later works, OmpF was refolded according to what
was to become the most classical procedure for b-barrel MPs (see
below), i.e. by transfer from urea to either lipid vesicles [43] or
detergent micelles [44].
The same year, the single-b-barrel outer membrane protein A
from E. coli (OmpA) was refolded by transfer from SDS to detergent
micelles [45], quickly followed by refolding from urea [46]. OmpA,
and its transmembrane domain, tOmpA (which lacks the periplasmic domain), were to become models of choice for MP folding
studies, which played a critical role in making possible the determination of tOmpA’s crystallographic [47,48] and solution NMR
[49] structures.
Organization of the table
With the stage thus set, we can turn to our table and try to
extract some lessons from it. It currently comprises 89 MPs, with
a slight excess of a-helical over b-barrel MPs. Unsurprisingly,
monomeric MPs are much more numerous than oligomeric ones,
but the number of the latter is far from negligible (cf. Fig. 1).
The table is organized as follows. MPs are listed in chronological
order, according to the year when successful (re)folding was first
published. If a given MP – the prototypical example being BR – has
been folded according to several different methods and in various
media, the most significant departures from the princeps protocol
are listed right after it, again in chronological order. In the few cases
where several relatively different but related MPs have been
(re)folded, as for proteorhodopsins in the case of BR, they are listed
right after the prototypical MP rather than at their chronological
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100
90
42
mono
aαmono
B
A
aαpoly
poly
mono
bβ mono
80
poly
bβ poly
29
Total
Total
Number of proteins
70
60
10
50
8
40
mono
aαmono
poly
aαpoly
mono
bβmono
poly
bβ poly
30
20
10
0
Year of first publication
Fig. 1. Number and types of integral membrane proteins that have been either refolded or folded de novo in vitro. MPs are distributed according to the secondary and
quaternary structure of their transmembrane domain: (i) monomeric a-helical MPs; (ii) oligomeric a-helical MPs; (iii) monomeric b-barrel MPs; (iv) oligomeric b-barrel MPs.
Each protein is counted only once, irrespective of the number of different ways it may have been (re)folded. In A, the cumulative number of MPs of each type that have been
(re)folded is plotted as a function of time, each protein being entered only once, on the year of the first successful report, even if it has been (re)folded using various methods.
In B, a histogram is shown of the total number of MPs of each type that had been (re)folded by at least one method at the end of 2013.
order. I have not attempted to draw too fine a distinction between
closely and more distantly related proteins, as it does not really bear
on the messages of the review.
The data in the table are then briefly analyzed with respect to
the types of MPs, the mode of production of the protein, the types
of denaturing and folding media, and the types of methods that
have led to successful in vitro folding. It is not my intent here, however, to dwell on folding mechanisms and steps, just to review
which approaches have succeeded, and for which proteins. Folding
in amphipols (APols) is just briefly mentioned here. It is discussed
more at length in another review in the same special issue [50].
The fact that so many chemically and physically different media
allow MPs to fold has interesting implications regarding the factors
that govern MP folding in general, a point that will be examined
elsewhere, in the discussion paper whose preparation launched
me on the present literature search [51].
Thirty-three years of folding attempts: an overview
Membrane proteins folded
A first interesting information that can be extracted from the
table is the overall number of MPs that have been folded or
refolded, what their types are (a-helical or b-pleated, monomeric
or oligomeric), and how their number has evolved over the years.
This is summarized in Fig. 1. After very slow beginnings, the number of novel MPs folded each year has kept steadily increasing
(Fig. 1A). Cumulated numbers reached only 4 ten years after Khorana’s princeps work, 23 twenty years later, and 89 by the end of
2013 (Fig. 1A), with constant acceleration. The latter is due in part
to the development of new methodologies for MP production,
including the uses of inclusion bodies (IBs) and of CFE, as well as
that of new folding media, including nanodiscs (NDs) and amphipols (APols) (see below). Broken down by types of MPs, 52 a-helical
and 37 b-barrel MPs have been (re)folded over the years, of which
10 and 8, respectively, are oligomeric proteins (Fig. 1B).
Origin of membrane proteins
The origin of MPs used for (re)folding experiments is summarized in Fig. 2. Historically, the first MPs to be refolded were naturally abundant MPs, such as BR, LHCs, or bacterial porins. Until
1990, all experiments kept to using MPs, whether overexpressed
or not, that had been produced in vivo and targeted to a membrane.
MPs were first purified in their native state, then denatured, and
then refolded. Some experiments involved refolding from fragments, with the view either of better understanding folding processes or of introducing labels. They have not been included here
(for reviews, see Refs. [52–54]).
The difficulties met by overexpression of MPs in a functional
form, which mostly derive from the limited volume afforded by
the plasma membrane of the host and the toxic effects of overloading the MP insertion machinery, pushed many experimenters to
explore the recourse to inclusion bodies (IBs), precipitates that
form in the cytosol when abundantly expressed MPs are not targeted to membranes. IBs are easy to purify and can yield tens of
mg of the target MP, but the latter is present under a misfolded,
aggregated state. Prior to folding, IBs are purified, washed with a
non-denaturing detergent such as Triton X-100, and then solubilized under denaturing conditions, usually with either urea or
SDS. Folding a MP from IBs appears to have been first described
in 1990 for LHCII [41], but the approach really took off after
1993–1994 (Fig. 2A). It has led, as of today, to the production of
42 folded MPs, that is nearly half of our total sample, with a
marked advantage to b-barrel MPs: 27 vs. 15 a-helical ones
(Fig. 2B).
First explored at about the same time with the b-barrel MP
PhoE [55], MP production by CFE has long remained in the background, but it has become widely used from 2003 on (Fig. 2A).
MPs obtained in vitro using CFE can be synthesized either in the
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J.-L. Popot / Archives of Biochemistry and Biophysics 564 (2014) 314–326
100
A
B
22
21
90
in vivo, no IB
in vivo, IB
80
CFE EC
CFE WG
13
Number of proteins
70
Total
60
6
50
6
4
4
2
5
3
3
2
2
1
40
aα
mono
mono
a αpoly
poly
bβ mono
mono
poly
βb poly
30
20
10
0
Year of first publication
Fig. 2. Origin of MPs that have been successfully (re)folded in vitro. Four types of origins are distinguished: (i) natural expression or overexpression in vivo, barring inclusion
bodies; (ii) in vivo expression in inclusion bodies; (iii) in vitro, cell-free expression in E. coli lysate (CFE EC); (iv) in vitro, cell-free expression in wheat germ lysate (CFE WG). In
A, the cumulative number of MPs obtained by each method that have been successfully (re)folded is plotted as a function of time, each protein being entered once for each
curve, on the year of the first successful report. Because some MPs have been (re)folded after being obtained by different methods and they are counted only once in the curve
summing all production methods, the latter shows less proteins than the sum of the individual curves. In B, a histogram is shown of the total number of MPs of each type that
have been (re)folded after being obtained by each method.
presence of a mild surfactant – lipid vesicles, NDs, detergent or
detergent-lipid mixed micelles, APols. . . –, in which case they are
supposed to insert and fold in the course of biosynthesis, or they
can be left to precipitate from a surfactant-free lysate. The precipitates thus obtained are much easier to dissolve than IBs and are
often solubilized using a non-denaturing detergent. In some cases,
however, they are dissolved either in SDS or in urea, bringing one
back to now classical folding procedures. E. coli lysates have
yielded by far the most folded MPs (28 of them), but since 2007
wheat-germ lysates are catching up (Fig. 2A). There are some outliers, like the use of a reticulocyte lysate supplemented with dog
pancreas lysosomes to express, fold and assemble a T cell receptor-CD3 complex [56]. CFE has been much more often used to
express a-helical than b-barrel MPs (32 vs. 4; Fig. 2B). There may
well be a circumstantial rather than a real technical basis for this
situation, however. On the one hand, b-barrel MPs appear particularly easy to fold from IBs, making it unnecessary, for most experiments, to turn to the much more costly and technically more
demanding CFE. On the other, the use of CFE has been strongly
boosted by the demand of biological and pharmaceutical research
for properly folded eukaryotic membrane receptors and channels,
which biases the distribution towards a-helical MPs.
Nature of the unfolded state
There are five main types of unfolded states from which
(re)folding is conducted (Fig. 3), plus some more exotic ones that
have been used only rarely. As already noted in H. Kiefer’s 2003
review [1], most a-helical MPs, whichever way they have been
unfolded, e.g. using organic solvents, or produced, e.g. as IBs, are
usually transferred to a strongly denaturing detergent, typically
SDS or LDS, and the latter exchanged for the renaturing medium.
GdnHCl has been used in a dozen cases, mainly for b-barrel MPs,
but far less frequently than urea. Urea, which, used alone, is usually
unable to keep a-helical MPs water-soluble (for a couple of exceptions, see Refs. [31,57,58]), has become the standard medium for
unfolding b-barrel MPs. Urea is sometimes used in combination
with SDS, but rarely [59–61]. The data in Fig. 3 are limited to the
cases where SDS, urea or GdnHCl have been used alone. To a large
extent, SDS is the medium of choice as a starting point for folding
a-helical MPs (20 of them), urea and occasionally GdnHCl for
b-barrel ones (30 and 12, respectively).
Less frequently used denaturing media from which folding has
been achieved without transfer to one of the above ones include
sarkosyl, mixed with digitonin, used as an intermediate to fold a
GPCR expressed in IBs [62]. Direct (re)folding from organic solvent
without first transfer to a denaturing surfactant appears exceedingly difficult, as it has not yielded a single routine protocol. It
may therefore be worth mentioning that refolding of BR has been
observed once upon transfer from trifluoroethanol (TFE; a solvent
that favors a-helix formation) to APols in the presence of retinal,
suggesting that the approach is feasible [63]. Despite multiple
attempts, however, conditions that permit to reproduce this observation could not be identified. Irreproducible results are generally
not mentioned in mainstream scientific literature (there is a specialized journal for them). In that case, however, chances that the
observation resulted from an experimental error are just nil, making it worth reporting. It is possible that the exact manner in which
the transfer of BO from formic acid/methanol to TFE is performed
be the source of irreproducibility.
An experimental situation that is becoming increasingly frequent is that the MP never exists as a full-length unfolded protein,
but is synthesized in vitro by CFE. As mentioned above, the growing
polypeptide chain can find itself in a surfactant-free aqueous
lysate, in which case it aggregates and precipitates, or it can fold
into and be kept soluble by a surfactant that has been added to
the lysate, be it detergent or mixed lipid/detergent micelles, lipid
vesicles, or NDs. More exotic surfactants include polymers, fluorinated surfactants, or amphipathic peptides (see below, Folding
media). Upon CFE in the presence of surfactants, folding is likely
to start during the synthesis, as is the case in vivo for most a-helical
MPs.
A special case of unfolding/refolding experiments is that
encountered during single-molecule force spectroscopy (SMFS).
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30
A
SDS
GdnHCl
25
Urea
CFE
SFMS
Number of proteins
20
15
10
5
0
a mono
α mono
a poly
b mono
α poly
b poly
β mono
β poly
30
α mono
B
α poly
25
β mono
β poly
Number of proteins
20
15
10
5
0
SDS
GdnHCl
Urea
CFE
SFMS
Fig. 3. Nature of unfolded states. Five cases have been distinguished: (i) MPs that have been denatured in SDS or LDS; (ii) MPs that have been denatured in GdnHCl; (iii) MPs
that have been denatured in urea; (iv) MPs that have been expressed in vitro; (v) MPs that have been unfolded by mechanical traction in an SMFS experiment, and shown to
have refolded after the traction was released. For each type of unfolded state, MPs are distributed as a function of the secondary and quaternary structure of their
transmembrane region, as defined in Fig. 1. In Panel A, MPs are grouped according to their structural type, in Panel B according to the nature of the unfolded state.
In these experiments, the MP, usually inserted in either a native or
a reconstituted membrane, is unfolded mechanically, by pulling
with the tip of an atomic-force lever on one of its termini, while
recording the traction needed to extract each sequence segment.
Upon releasing the traction, the protein reinserts and refolds. Reextracting the protein while measuring again the force profile
and comparing it to the original one makes it possible to determine
whether refolding has indeed occurred. First applied to an a-helical
sodium-proton antiporter [64,65] and to BR [66], the approach was
later extended to b-barrel MPs [67,68].
Folding media
The most frequently used folding media are the following: (i)
detergent micelles, or mixed lipid/detergent micelles; (ii) lipid
bilayers, in the form either of lipid vesicles or of NDs; (iii) APols
or related polymers (Fig. 4A). Detergents and mixed micelles provide the bulk of current data, followed by lipid vesicles and NDs.
NDs, to date, have only been used for MP production by CFE (see
e.g. Refs. [69–74]). In principle, it ought to be possible to identify
conditions under which scaffolding proteins, lipids and an
unfolded MP are all dissolved in a denaturant, presumably SDS,
and MP folding initiated concomitantly with the assembly of the
NDs. This experiment has not been described, however, and scattered unpublished information suggests that finding appropriate
conditions may not be easy.
APols appear as a promising medium for folding MPs. Initial
experiments were carried out on model proteins, BR and two
monomeric b-barrel MPs [75]. They were later extended to six
GPCRs [76–79] (reviewed in Refs. [77,80]) and two more b-barrel
proteins [81]. Transfer is effected from SDS in the case of a-helical
MPs, from urea for b-barrel ones. Non-anionic APols [78] and the
amphipathic polymer NVoy (or NV10) [82,83] have been successfully used for CFE, as well as SMALPs (particles comprised of a styrene/maleic acid copolymer and lipids, not unlike NDs) [19]. Why
APols appear as a particularly efficient medium for folding such
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J.-L. Popot / Archives of Biochemistry and Biophysics 564 (2014) 314–326
60
Detergent +
+/- lipids
22
+
B
A
Vesicles, NDs
50
Amphipols
Others
Total
Number of proteins
40
12
11
7
7
30
4
3
2
2
2
1
20
mono
aα mono
2
1
poly
aαpoly
mono
bβ mono
βb poly
10
0
Year of first publication
Fig. 4. Environment in which (re)folding takes place. Four types of environments have been considered: (i) detergent or mixed lipid/detergent micelles; (ii) lipid bilayers, in
the form of either lipid vesicles or NDs; (iii) APols and other amphipathic polymers; (iv) other media. In A, the cumulative number of MPs that have been successfully
(re)folded in each medium is plotted as a function of time, each protein being entered once for each curve, on the year of the first successful report. Because some MPs have
been (re)folded in different media and they are counted only once in the curve summing all folding media, the latter shows less proteins than the sum of the individual curves.
In B, histograms are shown of the types of media that have been used for the (re)folding of each structural type of MP.
fragile MPs as GPCRs is likely related, at least in part, to their stabilizing effect on MPs. A discussion of this point is beyond the goals
of the present survey and will be found in specialized reviews
[50,84,85].
More rarely used media include amphipathic peptides [86] and
fluorinated surfactants [18,87].
Modes of transfer
Modes of transfer include dilution, such as that of an SDSsolubilized a-helical MP into a non-denaturing detergent or lipid/
detergent mixed micelles, or that of a urea-solubilized b-barrel
MP into either micelles or a suspension of lipid vesicles. Denaturing detergents can also be removed by adsorption onto polystyrene
beads or by dialysis, diluted below their cmc, or precipitated as an
insoluble salt. For urea-denatured MPs, dilution is generally used,
either simultaneously for the whole preparation, or by injecting
slowly the urea solution into a solution of whichever surfactant
will act as the (re)folding medium.
Two factors are particularly worth paying attention to: (i) which
succession of media is the protein going to experience upon moving from a denaturing to a presumably renaturing environment;
and (ii) what is the concentration of proteins in the process of
refolding at a given time. Starting with the second point, it is intuitive that the higher the concentration of unfolded or partially
folded proteins at a given time, the higher the chances that
non-productive intermolecular interactions will get in the way of
productive intramolecular ones. Ways to prevent or limit the formation of intermolecular interactions include (i) to slowly inject
the solution of unfolded MP into the folding environment, so that
only a small fraction of the protein will be in the process of folding
at any given time (e.g. Ref. [88]; cf. Ref. [89], where this approach is
applied to soluble proteins or extramembrane MP domains); (ii) to
provide a large volume of non-denaturing surfactant, so as to dilute
the proteins (e.g. Ref. [23]); and (iii) to immobilize the unfolded
proteins onto a solid support, and to renature them before letting
them loose, so as to prevent (re)folding proteins from interacting
one with another (e.g. Refs. [62,90]).
As regards the question of how progressive the transition from
one type of medium to another should be, my personal view is that,
as a general rule, the more rapid the transfer is, presumably the
better. The reasoning is that a ‘‘semi-denaturing’’ environment,
such as that provided by a mixture of SDS and lipids from which
the SDS is slowly removed by dialysis, may have a greater chance
of inducing misfolding than a brutal transfer from a mostly SDS
to a mostly lipidic environment, because, at any intermediate point
along the way, the protein may adopt a conformation that corresponds to a free-energy minimum in that particular environment,
but is not native, and from which it may have difficulties to reemerge. This is what led us, when confronted with the challenge
of refolding BR in the presence of a minimum amount of lipids,
so as to reform 2D crystals, to introduce dodecylsulfate precipitation by K+ ions as a way to very rapidly move from one medium
to another [91]. This avoided introducing a large amount of lipids,
as is done in the dilution protocol [23]. Dodecylsulfate precipitation has subsequently been used for renaturing BR obtained by
CFE and transferred to SDS [92], and for transferring to detergent
micelles LHCII obtained by CFE and transferred to LDS [42]. It has
proven highly successful for folding a-helical MPs in APols
[63,75,76,78,79,93]. In comparative experiments in which
SDS-denatured BR was transferred to APols using either precipitation, dilution, dialysis, or adsorption onto polystyrene beads,
precipitation consistently gave the best folding yields [63,94]. It
would be desirable to explore this question more systematically
using a variety of MP models.
Helpers
Needless to say, the presence during (re)folding of molecules
that bind preferentially to the native state can improve folding
yields. By associating to fully or partially folded proteins whose
binding sites have adopted a native-like fold, they stabilize them
and steer folding towards the native state. They may also help
J.-L. Popot / Archives of Biochemistry and Biophysics 564 (2014) 314–326
321
Table 1
Integral membrane proteins that have been folded or refolded in vitro from a fully denatured state or obtained by cell-free expression. MPs are arranged in the chronological order
of the first demonstration of in vitro folding. Only the princeps papers are mentioned, except when subsequent papers are much more documented, resort to folding media or
conditions that are substantially different from the princeps ones, or otherwise contribute to the present discussion. In such cases, additional references concerning a given MP are
listed in chronological order following the princeps one. Inclusion in the table implies that at least a fraction of the folded material had demonstrated functionality and/or nativelike structure (NMR and/or diffraction data, oligomerization, native-like migration upon SDS–PAGE, cycles of unfolding/refolding by SMFS etc.; CD data alone, although suggestive,
have been considered insufficient as a demonstration of folding to the native state). Transmembrane structures are indicated with the following code: n-b: an n-strand b-barrel; na: an n-a-helix bundle; m n-b: an m-mer of n-strand b-barrels, etc. Transmembrane a-helical structures are on a pinkish background, b-barrel ones on a greenish one,
oligomeric structures on a darker background. The most frequently used folding media are indicated by a bluish background (detergents and detergent/lipid mixtures), a
brownish one (lipid vesicles or membranes, nanodiscs) or a purplish one (APols). Less frequently used folding media are on a greyish background. Abbreviations: 2D, twodimensional; APol, amphipol; CFE-EC, cell-free expression using an Escherichia coli lysate; CFE-WG, CFE using a wheat germ extract; DDM, n-dodecyl-b-D-maltoside; EIB-EC,
expression as inclusion bodies in E. coli; GdnHCl, guanidinium chloride; LDS, lithium dodecylsulfate; SDS, sodium dodecylsulfate; SMFS, cycles of unfolding/refolding by singlemolecule force spectroscopy; TM, transmembrane. ‘‘Detergent’’ refers to non-denaturing detergents (i.e., barring SDS, LDS and sarkosyl). Overlooked when preparing the table and
not included in the analyses is Ref. [184]. For general reviews, see e.g. Refs. [2,4,8,9,12,15,17,99–101].
322
Table 1 continued
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J.-L. Popot / Archives of Biochemistry and Biophysics 564 (2014) 314–326
preventing re-denaturation of proteins that have reached the
native state but are only marginally stable and can progressively
drift into non-native, e.g. aggregated states.
This role can be played by physiological ligands, but also by lipids. High-resolution crystallographic structures of MPs show lipids
bound to specific cranks of the protein transmembrane surface, as
well as in protein-buried sites (see e.g. [95,96], and references
therein). One can visualize that, as such sites form while the refolding protein explores conformations, those conformations that bind
lipids are stabilized, steering the folding process in the right direction. Another, related factor may be that, by binding to the reforming transmembrane surface, lipids displace detergent molecules,
and prevent those from interfering with the formation of tertiary
structure interactions. As an example, GPCRs interact with and
323
are stabilized by cholesterol [97,98], which has led to using cholesterol hemisuccinate as a helper in folding protocols [78]. Comparative experiments in which BR and several GPCRs were folded in
APols have repeatedly shown a small, but significant increase in
folding yield (e.g. from 80 to 90% for BR, or from 60 to 70%
for GPCRs) when APols are supplemented with lipids (typically in
a 5:1 APol/lipid mass ratio) [63,76].
Oligomerization
Folding oligomeric MPs is a particularly challenging process, but
it has been achieved in quite a few cases (Fig. 1B), both for a-helical
(LHCII, DAGK, a T cell receptor-CD3 complex, EmrE, MscL, KvAP,
Erb3 transmembrane domain and the Shaker channel) and for
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b-barrel MPs (8 homotrimeric porins). For the latter, the folding
protocol always relies on transfer from urea to either detergent
micelles or lipid vesicles, except for LamB, which was refolded by
transfer from GdnHCl to detergent micelles (Table 1). For a-helical
oligomers, both the complexity of the structure and the methods
used are much more variable, CFE and/or transfer from SDS being
the most frequent approaches (Table 1). The special problems
raised by folding oligomeric MPs are further evoked below.
was elusive as long as conditions had not been found to transfer
each fragment from organic solvent to SDS without generating
any aggregation, as controlled by SDS–PAGE (unpublished
observation).
Finally, the variety of media in which MPs can reach a nativelooking, functional 3D structure should give one reasons to pause
when considering the constraints from the membrane environment
that are supposed to guide MP folding. This thought-provoking
point will be discussed elsewhere [51].
Conclusion
Acknowledgments
From this rapid survey, one can draw a number of conclusions.
First, success is not as elusive as is often thought. Folding MPs
in vitro requires care, attention and perseverance, but it is far from
the very long shot, better left to others, that it is often thought to
be, and the probability of success is far from negligible. The body
of acquired experience is now rich enough to provide guidelines
as to what is most likely to succeed and what should be regarded
as less solidly validated. A particularly clear lesson, for instance,
well known to practitioners, is that a-helical MPs are best folded
starting from the SDS-solubilized form, b-barrel MPs from the
urea-solubilized one. As regards the method of transfer, my personal bias, which seems at least to some extent supported by the
facts, is that a very rapid transfer to the renaturing medium, such
as by dodecylsulfate precipitation or quick, extensive dilution of
urea, may be preferable to slow methods such as dialysis or SDS
adsorption onto BioBeads, the rationale being that the refolding
protein does not experience a variety of partially denaturing conditions, some of which may favor misfolding. There is, however, a
dearth of systematic experiments to validate, disprove or qualify
this intuition.
Second, hard as it may seem, folding oligomeric MPs can be
made to work. The challenge here is that, as for the folding of
monomeric MPs, folding conditions must favor intramolecular over
intermolecular interactions at the step of folding the monomer, so
as to prevent improductive aggregation, but at the same time
newly formed, often unstable monomers must be given a chance
to correctly assemble before misfolding and/or aggregating. It
may well be that in some cases the completely folded monomer
is not the most stable form as an isolated entity, and complete folding occurs only upon interaction with other monomers. One thinks,
for instance, of MPs like OmpF or some channels, whose final fold
includes regions that are not extended or hydrophobic enough to
span a lipid bilayer without being protected by the other subunits
from contact with lipid hydrophobic chains. Unexpected folding
and assembly intermediates will undoubtedly be discovered, such
as the asymmetrical dimers of OmpF that form on its way to trimerization [44]. One can expect that the search for folding environments that be as mild as possible, so as to prolong the life of
the unassembled monomer, will be critically important in this case
of figure, and the protein concentration at which folding is conducted a major variable.
Third, there is more than one way to skin a cat. When mainstream approaches do not succeed, it may be useful to review
the variety of media and techniques that have been shown to work
with other MPs and ponder whether they could not be adapted to
the protein at hand. Great attention should be paid to the starting
point, namely the unfolded protein. In my personal experience,
those folding attempts that failed could often be traced to improper starting material: a preparation in which all intermolecular
interactions have not been eliminated, for example, or in which
the supposedly unfolded MPs may have retained or formed some
extent of tertiary structure, which may not necessarily be nativelike, is unlikely to give high folding yields. In refolding BR in lipids
starting from chymoptryptic fragments, for instance [91], success
I am deeply grateful to Jacqueline Barra for her invaluable help
with collecting the literature and preparing the figures, as well as
to Jean-Louis Banères, Jim Bowie, Susan Buchanan, Laurent Catoire,
Tassadite Dahmane, Mark Dumont, Don Engelman, Karen Fleming,
Jörg Kleinschmidt, Daniel Müller, Daniel Otzen, Francesca Zito and
Manuela Zoonens for helping me to plug holes in Table 1 and/or for
commenting on early drafts of the review.
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