Membrane protein folding makes the transition

Paula J. Booth
a,1
and Jane Clarke
b,1
a
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom; and
b
Department of Chemistry and Medical Research Council Centre for Protein Engineering, University of Cambridge, Cambridge
CB2 1EW, United Kingdom
T
he study of the folding of mem-
brane proteins has lagged far
behind that of small soluble
proteinsyet proteins that
reside within biological membranes ac-
count for approximately a third of all
proteomes. The article by Huysmans et al.
in this issue of PNAS (1) represents a
breakthrough by reporting a compre-
hensive -value analysis of the folding of a
membrane protein (i.e., PagP) into a
lipid bilayer. -value analysis is the most
powerful tool for experimental analysis
of protein folding pathways (2). It com-
bines protein engineering with equilibrium
and kinetic measurements to determine
which regions of a protein are largely fol-
ded (high -values) or largely unfolded
(low -values) at the rate-limiting
transition state.
There are two major structural classes
of integral membrane proteins: -helical
bundles and -barrels. The latter are found
in the outer membranes of Gram-
negative bacteria and mitochondria,
whereas helical structures are ubiquitous,
occurring, for example, in plasma and in-
ner membranes. PagP has a -barrel
structure and resides in the outer mem-
brane of Escherichia coli. The analysis
presented by Huysmans et al. (1) sheds
light on the transition state structure for
formation of this -barrel in a bilayer. The
work complements a previous -value
study of membrane protein folding that
was the rst to map out a membrane
protein transition state, but in this case, for
folding of an -helical protein, bacterio-
rhodopsin, into lipid-detergent mixtures
(3). Thus detailed insight into the folding
mechanisms of the two major membrane
protein structural classes is now emerging.
A key feature of this current study (1) is
the examination of folding into a lipid bi-
layer. To achieve this, Huysmans et al. (1)
exploited a previously demonstrated trait
of -barrel proteins: they can be reversibly
refolded from a urea-denatured state into
lipid bilayer vesicles, and the kinetics and
thermodynamics of the folding reaction
can be determined (4, 5). The successful
demonstration of this for PagP (6) paved
the way for a -value study. Identication
of conditions for reversible folding of PagP
relied on manipulation of the lipid to
protein ratio. At low ratios, PagP folding
was protein concentration-dependent, but
at the lipid-to-protein ratio used here
(3,200:1), refolding was completely rever-
sible and protein concentration
independent. The folding and unfolding
kinetics were determined, and, perhaps
surprisingly, conditions could be found
(urea concentrations greater than approx-
imately 8 M) in which folding could be
characterized as a simple two-state re-
action. It is also of note that the choice of
a -barrel protein means that folding can
be studied from an apparently fully un-
folded, denatured state. Most helical
membrane proteins retain signicant re-
sidual structure in common denaturants
such as urea and SDS, but the denatured
state of PagP in 10 M urea, although as-
sociated with the bilayer, is essentially
unstructured (as judged by circular di-
chroism and uorescence spectra). The
use of urea gives added importance to the
PagP work, as urea apparently does not
partition into the bilayer, unlike SDS used
for studies of the folding of several
-helical proteins.
Nineteen variants of PagP were inves-
tigated, at least one mutation in each
-strand, thus giving a snapshot across the
entire protein. The protein folds via a
polarized transition state with the barrel
partly formed and the C-terminal part of
the protein signicantly more structured
than the N-terminal regions. The authors
propose that the results are consistent with
a tilted folding-insertion mechanism (Fig.
1), as has been seen in simulations for in-
sertion of folded OmpA into a bilayer (7).
The results also agree with previously
proposed models of concerted folding and
insertion of -barrels into membranes (8);
as an unfolded barrel protein within a bi-
layer is unlikely, as it would expose po-
tential hydrogen bonding groups (9).
Helical membrane proteins, by contrast,
seem to fold by a fundamentally different
mechanism than barrels (9, 10). Individual
transmembrane helices can be stable en-
tities in bilayers, as backbone hydrogen
bonding is satised locally within the helix.
Fig. 1. Schematic diagrams of proposed folding models for -barrel and -helical membrane proteins,
highlighting potential transition state structures from -value studies. Folding of a -barrel protein oc-
curs from a fully denatured, membrane-absorbed state in urea with a tilted, partly inserted transition
state as proposed by Huysmans et al. (1). In contrast, folding of an -helical protein such as bacterio-
rhodopsin occurs from a partly denatured state in SDS, which contains some helical content. The tran-
sition state is proposed to have signicant native helix content. Only one transmembrane helix has been
analyzed by -values (3), and this suggests a largely formed helix with partial helix formation at the
cytoplasmic side (shown in the bottom of this diagram, with this helix outlined in black). The degree of
structure in other helices is estimated from previous studies (e.g., ref 15). Unfolded structure is shown in
red and folded structure in blue. SDS is shown in green. PagP was folded into lipid bilayers vesicles,
whereas bacteriorhodopsin was folded into mixed DMPC/CHAPS micelles (the detergent CHAPS is shown
here capping the edges of a DMPC disc).
Author contributions: P.J.B. and J.C. wrote the paper.
The authors declare no conict of interest.
See companion article on page 4099.
1
Towhomcorrespondence may be addressed. E-mail: paula.
booth@bristol.ac.uk or jc162@cam.ac.uk.
www.pnas.org/cgi/doi/10.1073/pnas.0914478107 PNAS | March 2, 2010 | vol. 107 | no. 9 | 39473948
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Thus partially folded states occur within a
membrane, as conceptualized by the clas-
sic two-stage folding model, whereby
helices rst form within a membrane (10).
Final helix packing and tertiary structure
formation then occurs as a second stage
(Fig. 1). Models emerging for in vivo
folding also propose this type of folding
mechanism, as transmembrane helices
form within the translocon complex
during cotranslational folding, and this is
followed by nal helix packing and folding
within the membrane (11, 12). Inves-
tigations of folding mechanisms by studies
in vitro are also consistent with the
overall two-stage idea, although the sit-
uation is more complex than two simple
steps (9, 13).
The increased hydrophobicity of helical
membrane proteins compared with
-barrels makes the former more chal-
lenging to experimental manipulation.
Consequently, the folding system used for
the bacteriorhodopsin -value study (3) is
more complex than the urea/bilayer one
used by Huysmans et al. for PagP (1). A
partially denatured state of bacterio-
rhodopsin in SDS was used; this contains
just more than half the native helix content
and seems to represent a critical helical
core that is a prerequisite for correct
folding. More extensive denaturation of
bacteriorhodopsin requires acidic organic
acids that are incompatible with refolding
in detergent or lipid systems. Two-state
reversible folding conditions were estab-
lished for bacteriorhodopsin using lipid/
detergent mixtures of 1,2-dimyristoyl-sn-
glycero-3-phosphocholine (DMPC) and
CHAPS that seem to form elongated mi-
celles or DMPC bilayer discs capped by
CHAPS detergent, referred to as bicelles.
Overall, these bilayer discs are not much
larger than the embedded protein, but
seem to provide a good bilayer mimic for
helical proteins. The SDS denaturant will
also partition; thus folding is studied in
DMPC/CHAPS/SDS mixtures. The fold-
ing process studied for bacteriorhodopsin
represents nal helix formation and pack-
ing, which is a key folding event for helical
proteins. -values were determined
throughout one helix of the seven-helical
bacteriorhodopsin. These revealed that
most of the helix is formed in the tran-
sition state, but intermediate and low
-values toward the cytoplasmic end of the
helix suggest only partial helix formation
and tertiary contacts. Intermediate -val-
ues can also arise from multiple transition
states or folding paths, which could be
envisaged for packing of stable helical
entities. Many intermediate -values are
also seen in the PagP study (1). The pos-
sible contributions of the bilayer environ-
ment and intermolecular forces to such
intermediate values await further exami-
nation for both protein classes.
Membrane proteins may reside within a
heterogeneous and largely hydrophobic
environment, which complicates exper-
imental study, but given careful consid-
eration of experimental conditions,
membrane protein folding can be inves-
tigated in detail, and many of the
approaches that have worked well for
soluble proteins can be suitably trans-
ferred. These successful applications of
-value analysis are especially propitious,
as they will reveal structures of key mem-
brane protein intermediates and transition
states. The membrane environment itself
directly affects protein folding (13), with
the membrane elastic properties inuenc-
ing the folding rates, yields, and pathways
of both helical and -barrel proteins.
Matching of the hydrophobic thickness of
the bilayer to the protein is also vital, with
a mismatch affecting the yield, coopera-
tivity, thermodynamics, and tilt of the
folding protein structure. This requires
comprehensive investigation of the lipid
properties. An excellent start has pre-
viously been made for -barrel proteins
using another E. coli outer membrane
protein, OmpA (4), in which lipids with a
hydrophobic thickness of approximately 30
were found to give the best match to the
protein. The lipids used for PagP are
shorter, fully saturated lipids (1, 6), which
have different elastic properties and
thicknesses than those used for OmpA. It
is interesting that different lipids give rise
to two-state reversible folding for two dif-
ferent -barrel proteins from the same
outer membrane. This further emphasizes
how diverse conditions may be needed to
probe the folding of apparently similar
structures. A full understanding of the in-
uence of the lipid bilayer will require
further investigation of several proteins
under contrasting lipid conditions, to-
gether with a quantitative understanding
of the relevant lipid properties.
In some respects, the structures of
membrane proteins, with only two main
structural classes, are generally less com-
plex than those of soluble proteins. Huys-
mans et al. (1) suggest that there might be
a generic tilted folding-insertion mecha-
nism for -barrel membrane proteins.
Conservation of folding mechanisms in
protein families has been observed before
particularly in all -proteins in which the
topology is complex (14). This lends fur-
ther support to the notion that there may
be general folding mechanisms for the two
folding classes: insertion and packing of
preformed helical elements or concerted
folding and insertion. Intriguingly, the
early events in the folding of the -barrel
proteins might also be more complexin
the study by Huysmans et al., the folding
kinetics of PagP became much more dif-
cult to interpret below 7.8 M ureasug-
gesting that early stable intermediates
might be populated.
The importance of this study and that of
bacteriorhodopsin is that they show it is
possible to investigate the folding mecha-
nisms of membrane proteins in detail
and to explicitly test simulations of their
folding. Neither system is trivial to work
on, but these studies (1, 3) pose a chal-
lenge to other experimentalists in the eld.
In studies of the folding of soluble pro-
teins, the assumption is that the folding
pathway in vitro is essentially the same as
pathway in vivo. Is the same true of
membrane proteins? It is clear that the
physiochemical properties of membranes
have an effect on the physical/chemical
properties of membrane proteins. These
two studies, which elucidate the mecha-
nism of the folding of membrane proteins,
open the door to experimental inves-
tigations of these complex questions.
ACKNOWLEDGMENTS. J.C. is a Wellcome Trust
Senior Research Fellow. P.J.B. thanks the Royal
Society for a Wolfson Research Merit Award.
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3948 | www.pnas.org/cgi/doi/10.1073/pnas.0914478107 Booth and Clarke