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INTRODUCTION |
A substantial proportion of newly synthesized proteins are
inserted into membrane bilayers, and studies on a variety of systems have shown that this process can occur co- or post-translationally. However, the post-translational targeting of hydrophobic membrane proteins is potentially problematic because these proteins are by
nature prone to aggregation in polar environments. Hence, considerable attention has centered on the mechanisms by which these proteins are
initially maintained in a soluble form, and how the hydrophobic regions
are then transferred from an aqueous milieu into the apolar regions of
the membrane bilayer. The majority of chloroplast and mitochondrial
membrane proteins, and probably some bacterial membrane proteins, are
inserted post-translationally, and studies on these systems have
demonstrated two broad categories of insertion mechanism, which can be
regarded as "assisted" and "unassisted" according to whether
protein translocation machinery is utilized (reviewed in Ref. 1).
In bacteria, the secretory
(Sec)1 apparatus, is used for
the translocation of proteins into the periplasm, and the same
apparatus is also required for the insertion of some membrane proteins. Additional factors are also used for the targeting of membrane proteins, particularly signal recognition particle (SRP) and FtsY (2-7). A similar assisted pathway operates in plant chloroplasts for
the targeting of some cytosolically synthesized thylakoid membrane
proteins. After import across the chloroplast envelope, a subset of
thylakoid membrane proteins, exemplified by the major light-harvesting
chlorophyll-binding protein, Lhcb1, are targeted by a pathway that
requires GTP, a stromal form of SRP and FtsY (1, 8-10). This pathway
is thus similar in many respects to bacterial SRP-dependent
pathways and is presumably inherited from the cyanobacterial-type
progenitors of plant chloroplasts.
Other thylakoid membrane proteins are targeted by a second pathway that
exhibits very different characteristics. Subunit II of the integral
CFo component of the ATP synthase (CFoII), and subunits X and W of photosystem II (PsbX, PsbW) are synthesized with
bipartite presequences in which the stroma-targeting peptide is
followed by a hydrophobic "signal" peptide. These signal peptides are similar in overall terms to Sec-type signal peptides that direct
the targeting of lumenal proteins across the thylakoid membrane by the
SecYEG complex. However, these proteins integrate into the thylakoid
membrane in the absence of stromal factors, nucleoside triphosphates,
or a
pH (11, 12). Furthermore, proteolysis of thylakoids, which
blocks Sec-, SRP- and the lumen-targeting
pH dependent mechanisms,
has no effect on the insertion of these proteins (13). In the absence
of any identifiable proteinaceous targeting factor, it has been
proposed that they may insert spontaneously into the thylakoid membrane.
This particular insertion mechanism is unusual because it
involves the use of cleavable signal peptides for unassisted membrane insertion (at the very least, SRP, FtsY, and Sec involvement can be
ruled out). Only one other protein, M13 procoat, has been shown to be
synthesized with an N-terminal signal peptide yet inserted into the
Escherichia coli plasma membrane by a Sec/SRP-independent mechanism (for review, see Ref. 14), although procoat insertion does
require the activity of a recently identified membrane-bound component,
YidC (15).
Irrespective of whether unidentified membrane-bound machinery is
required for the insertion of the above thylakoid membrane proteins,
these proteins represent simple and attractive model systems for the
analysis of the early stages of the insertion process. The final stages
of the pathway have been characterized to some extent: the two
hydrophobic regions (one in the presequence and the other in the mature
protein) insert into the membrane, flipping the central acidic region
across the membrane and thereby forming a loop intermediate structure.
Cleavage by thylakoidal processing peptidase on the trans
side of the membrane releases the presequence, leaving the N terminus
of the mature protein in the thylakoid lumen. The formation of the loop
structure has been confirmed (16), but the early stages of the
insertion pathway are very poorly understood and the same applies to
most other membrane proteins. The basic problem is identical in nearly
every case: membrane proteins are inherently difficult to prepare and characterize in aqueous solvents and must normally be stabilized by
detergents or chaotropic agents.
The relatively simple insertion mechanism used by the PsbW-type
proteins, together with the notable insertion-competence of the
in vitro translation products, suggested that these proteins may be inherently stable in aqueous phases and hence good model systems
for the study of membrane proteins in general. We describe here the
purification of pre-PsbW from an E. coli overexpression system in high yields and its characterization in aqueous solution and
membrane-mimicking environments.
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EXPERIMENTAL PROCEDURES |
Overexpression and Purification of Pre-PsbW--
The coding
sequence for spinach pre-PsbW (17) was cloned into the pMW172 vector
for overexpression in the C41 strain of E. coli (18).
Expression was induced by the addition of 1 mM isopropyl
-D-thiogalactoside, and the cells were cultured
overnight. Cells were harvested, washed with 50 mM
Tris-HCl, pH 8, and resuspended in 20% sucrose, 50 mM
Tris-HCl, pH 8.0. The cells were lysed by passing them twice through a
French press at 1000 p.s.i. The inclusion bodies were collected by
centrifugation at 5000 × g for 10 min through a 40%
sucrose pad (wash A), and the pellet was washed with buffers B, C, and
D (buffer B = 0.4 M NaCl, 1 mM
dithiothreitol (DTT), 0.5% Nonidet P-40, 10 mM Tris-HCl,
pH 8.0, 0.1 mM EDTA; buffer C = 50 mM
NaCl, 1 mM DTT, 0.5% Nonidet P-40, 10 mM
Tris-HCl, pH 8.0, 0.1 mM EDTA; buffer D = 50 mM NaCl, 1 mM DTT, 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). Finally, the pellet was resuspended
in 8 M urea, 30 mM Hepes/KOH, pH 8.0, 1 mM DTT, 0.1 mM EDTA and left to rotate slowly
overnight at 10 °C. Inclusion bodies were collected by
centrifugation at 18,000 × g, resuspended in the same
buffer, and left rotating at 10 °C for at least 2 h. The
preparation was centrifuged again at 18,000 × g and
resuspended in PGEDS buffer (50 mM sodium phosphate buffer,
pH 8.0, 5% (w/v) glycerol, 1 mM EDTA, 1 mM
DTT, 50 mM NaCl) and left rotating at 10 °C for 1 h. The purified inclusion bodies were collected by centrifugation at
18,000 × g and solubilized in PGEDS buffer including
Sarkosyl (2% v/v) and left to rotate slowly overnight at 10 °C.
Purified solubilized protein was dialyzed against 20 mM
Tris-HCl, pH 8.0, 1 mM DTT to remove free Sarkosyl.
Overexpression and purification were analyzed by Tricine
SDS-polyacrylamide gel electrophoresis (19) and Western blotting.
Size Exclusion Chromatography of Purified Pre-PsbW--
A
Superose 6 gel filtration column (HR 10/30, Amersham Pharmacia Biotech)
was equilibrated in 20 mM Tris-HCl, pH 8.0, 1 mM DTT. 200 µl of dialyzed pre-PsbW was loaded onto the
column, which was then run at 0.2 ml/min. Fractions were analyzed by
Tricine SDS-polyacrylamide gel electrophoresis. Using identical
conditions, the same column was calibrated using blue dextran (2000 kDa), ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa),
ovalbumin (43 kDa), and RNase (13.7 kDa). Fractions containing the
pre-PsbW were pooled and concentrated using Amicon centriprep
concentrators with a YM3 filter. The sample was incubated in 4 M guanidinium hydrochloride (GuHCl) containing 1 mM DTT and applied to the Superose 6 column
(pre-equilibrated and eluted with 20 mM Tris-HCl, pH 8.0, 1 mM DTT). Fractions were analyzed using Tricine
SDS-polyacrylamide gel electrophoresis. For studies in detergent
micelles, SDS was added to the protein sample to a final concentration
of 0.5% (w/v). The mix was left at room temperature to equilibrate for
30 min before analysis.
Circular Dichroism Measurements--
Far UV circular dichroism
spectra were acquired for pre-PsbW at a concentration of 10 µM. Circular dichroism spectra were recorded on a Jasco
J-715 spectropolarimeter, using a quartz cell with a 1-mm path length.
Typically, a scanning rate of 100 nm/min, a time constant of 1 s,
and a bandwidth of 1.0 nm were used. The spectral resolution was 1.0 nm, and 16 scans were averaged per spectrum. All spectra had the
background obtained with buffer alone subtracted from them. Structure
fitting was carried out using the cdsstr program as described in Refs.
20 and 21.
Fluorescence Spectroscopy--
Fluorescence emission spectra
were obtained for pre-PsbW at a concentration of 3 µM.
Experiments were performed on a Perkin-Elmer LS50 luminescence
spectrometer. Tryptophan fluorescence was measured using an excitation
wavelength of 280 nm. Excitation and emission bandwidths were both 4 nm. Typically, 16 scans were averaged for each spectrum. All spectra
had the background obtained with buffer alone subtracted.
Purification of Near Mature-size PsbW--
Samples of 20 µM pre-PsbW in SDS were passed through a NAP-10 column
(Amersham Pharmacia Biotech) containing 20 mM Tris-HCl, pH
8.0, 1 mM DTT, 5%
n-octyl-
-D-glucopyranoside, and the eluate was mixed with 50 units of immobilized trypsin (Pierce) in the same
buffer. The digestion mix was rotated slowly at room temperature for
3 h, spun to remove the trypsin beads, and the mixture applied to
a mono-Q column (Amersham Pharmacia Biotech). The column was washed
using 100 ml of 20 mM Tris-HCl, pH 8.0, 1 mM
DTT, 2% n-octyl-
-D-glucopyranoside and
eluted in the same buffer with a gradient of 0-500 mM
NaCl. Elution of the PsbW degradation product was monitored by
immunoblotting and peak fractions containing the protein were exchanged
from n-octyl-
-D-glucopyranoside into SDS by
passing through a Nap-10 column pre-equilibrated in 20 mM
Tris-HCl, pH 8.0, 1 mM DTT, 0.5% SDS. Protein
concentrations were determined by measurement of the
A280 and A260 after
subtraction of the background obtained with the buffer alone. Protein
concentration was obtained using a correction formula (+): 1.55 × A280
0.76 × A260 = mg of protein/ml (22).
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RESULTS |
Overexpression and Purification of Pre-PsbW--
The goal of this
study was to analyze the structures of pre-PsbW and mature PsbW to
obtain information on key points of the overall insertion pathway. The
single most important objective was to prepare the precursor protein in
aqueous solution because the early stages of membrane protein insertion
are so poorly understood. Here it is important to point out that,
although the first "envelope transit" signal in the presequence is
probably removed prior to insertion into thylakoids, the full precursor
protein is fully competent for thylakoid insertion and has been
routinely used as a substrate in the in vitro insertion
studies carried out to date (12, 16). This form of PsbW was chosen for
biophysical analysis because the hydrophilic nature of the envelope
transit domain was predicted to help maintain the protein in a
buffer-soluble form. In addition, data on the full precursor protein
are valuable because they provide insights into the structure of the
protein prior to transport into the chloroplast.
Spinach pre-PsbW (molecular mass 14 kDa) was over-expressed in E. coli as detailed under "Experimental Procedures" and the protein was observed to form inclusion bodies, which were purified through several wash steps (Fig.
1A, lane IB). These
inclusion bodies were found to be insoluble in urea, and the pre-PsbW
was effectively purified to near homogeneity by simply washing the inclusion bodies twice in urea and then once in buffer, thereby solubilizing and removing the majority of contaminating proteins. Pre-PsbW remained in the insoluble pellet fraction and was then solubilized using the detergent Sarkosyl; Fig. 1B shows that
the majority of protein is found in the supernatant fraction when this
suspension is centrifuged at high speed. This procedure yields pre-PsbW
in a purified form and the identity of the protein was confirmed by
Western blotting using antibodies raised against a C-terminal region of
spinach PsbW (Fig. 1, A and B, lower
panels).

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Fig. 1.
Purification and solubilization of inclusion
bodies containing pre-PsbW. A, expression of spinach
pre-PsbW was induced in E. coli and samples analyzed of the
uninduced and induced cells (PI, I), the pellet
and supernatant fractions after cell lysis (Pel,
S/N), four wash steps (A-D, see
"Experimental Procedures"), and the final inclusion body
preparation (IB). B, the inclusion bodies were
washed twice in urea and once in buffer, and the pellet and supernatant
fractions analyzed. The pellet from the buffer wash was then
solubilized in Sarkosyl and centifuged to yield a pellet fraction and
solubilized pre-PsbW in the supernatant (S/N).
Samples were analyzed using Tricine-SDS-polyacrylamide gel
electrophoresis and Coomassie staining (top panels) or
immunoblotting using an antiserum raised against spinach PsbW
(bottom panels). Molecular weight markers are shown on the
left side of the gel (in kDa).
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The Sarkosyl-solubilized pre-PsbW was extensively dialyzed against Tris
buffer/DTT to remove free detergent, and the sample was subsequently
spun at 100,000 × g to remove any insoluble
aggregates. Unexpectedly, the majority of the pre-PsbW remained fully
soluble in Tris buffer, and we characterized this protein using
calibrated gel filtration chromatography (using Superose 6). The
elution profile (Fig. 2, dotted
line) shows that the protein elutes in fractions 15-30,
indicative of high molecular mass multimers (average size calculated to
be 180 kDa). However, these fractions were combined, concentrated, and
unfolded in the presence of GuHCl and then re-run on the gel filtration
column. Under these conditions the protein elutes primarily as a sharp
peak (fraction 39) corresponding to a molecular mass of about 25-30
kDa (Fig. 2, solid line). The column was pre-equilibrated
and eluted in Tris buffer containing DTT, and the GuHCl was found to
elute much later (fractions 42-45; not shown). Thus, pre-PsbW can be
prepared in a Tris-soluble form in a size indicative of a monomer or
dimer. The protein is purified to homogeneity as judged by
silver-staining of polyacrylamide gels (not shown).

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Fig. 2.
Purification of Tris-soluble pre-PsbW.
The solubilized pre-PsbW (see Fig. 1B) was dialyzed against
Tris buffer/DTT to remove Sarkosyl (see "Experimental Procedures")
and chromatographed on a Superose 6 gel filtration column in the same
buffer; the elution profile is denoted by the dotted line.
Fractions containing pre-PsbW were pooled, concentrated, incubated with
4 M GuHCl, and re-chromatographed on the same column
(solid line).
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Biophysical Analysis of Pre-PsbW--
The formation of
-helices
is a key event during membrane protein insertion. Energetic
considerations strongly suggest that these must form prior to full
insertion (for review, see Ref. 23), but the precise timing of
-helix formation is unclear in most cases. In principle, these
structures may form in solution or they may form preferentially in the
membrane interface region where competition for H-bond formation by
water molecules may be reduced. In most cases this problem is extremely
difficult to address because of the insoluble nature of recombinant
membrane proteins in aqueous buffer. The Tris-soluble forms of pre-PsbW were therefore subjected to biophysical analysis to probe their structures in more detail.
The far UV circular dichroism spectrum of the initial, multimeric
Tris-soluble form of pre-PsbW (average size 180 kDa) indicates a high
proportion of
-helical structure characterized by negative maxima at
208 and 222 nm. This secondary structure is predictably lost upon
unfolding in the presence of GuHCl (Fig.
3A). The second Superose
eluate (monomer/dimer) exhibits, by comparison, a substantial loss of
secondary structure producing a relatively flat spectrum, which
indicates the presence of very little conserved secondary structure
(Fig. 3B). We deduce from these data that Sarkosyl only partially solubilizes the inclusion bodies containing pre-PsbW, forming
multimeric structures that the GuHCl is then able to fully unfold. The
addition of SDS above the critical micelle concentration leads to a
rapid formation of
-helical structure (Fig. 3B), and we
conclude that the Tris-soluble "final preparation" is probably unable to form
-helices because of competition from water molecules for H-bonding.

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Fig. 3.
Circular dichroism spectroscopy of pre-PsbW
in aqueous buffer and SDS micelles. A, analysis of a
multimeric form of pre-PsbW in Tris/DTT buffer (1st eluate from
Superose 6 column described in Fig. 2). The figure shows the far UV
circular dichroism spectra of pre-PsbW in Tris/DTT (solid line), and in
the presence of 4 M GuHCl (dashed line), both at
a protein concentration of 10 µM. The unfolded spectrum
is only plotted to 210 nm as all the photons below this point are
absorbed by guanidinium hydrogen chloride. B, far UV CD
spectra of the pre-PsbW following GuHCl treatment and gel filtration
(second Superose 6 eluate shown in Fig. 2). Spectra are shown for the
protein in Tris/DTT (solid line) and after incorporation
into SDS micelles (dashed line).
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The presence of substantial
-helical structure in the initial
multimeric preparation of pre-PsbW suggests that these
-helices may
have been able to form in the relatively hydrophobic interior of the
larger structure. Pre-PsbW contains two tryptophan residues, both in
the transmembrane region of the mature protein, and the tryptophan
emission spectrum of the multimeric pre-PsbW also indicates a folded
structure. Fig. 4 shows that the emission
spectrum of the multimer exhibits a peak at 330 nm that is
characteristic of tryptophan residues within a hydrophobic environment
(solid line). The fluorescence emission peak becomes
red-shifted to 350 nm upon addition of GuHCl, indicating exposure of
the tryptophans to a hydrophilic environment (dashed line)
as expected when the protein unfolds. In contrast, the fluorescence
emission profile of the monomer/dimer in Tris buffer
(circles) shows a peak at 336 nm, red-shifted 6 nm from that
of the folded multimer. When GuHCl is added to the dimer, a spectrum
similar to the unfolded multimer is observed. These data suggest that
the monomeric/dimeric protein is not as tightly folded as the multimer,
but they also suggest that the tryptophan residues are not fully
exposed to aqueous solvent and that the protein may therefore be folded
to some extent, despite lacking conserved secondary structure.

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Fig. 4.
Tryptophan fluorescence characteristics of
pre-PsbW. Fluorescence emission spectra of "multimeric"
pre-PsbW in Tris/DTT (first Superose eluate; solid line),
and in the presence of 4 M GuHCl (dashed line),
and of the second Superose eluate (containing monomeric/dimeric
pre-PsbW) in Tris/DTT (circles) or GuHCl
(crosses).
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Secondary Structure Analysis of the Mature Protein--
The above
studies indicate that pre-PsbW adopts an
-helical structure within
SDS micelles, but these data do not indicate whether both hydrophobic
regions contribute to the observed spectrum. To gauge the structure of
the detergent-solubilized precursor more precisely, we sought to
compare it with that of the mature protein, which lacks the signal
peptide. A near mature-size protein can be generated from pre-PsbW by
trypsin digestion because the mature protein contains only a single
basic residue, arginine, at position 5. Fig.
5A shows that trypsin cleaves
the majority of pre-PsbW to this "mature" size after 30 min of
digestion. Most of the pre-PsbW disappears by this time point although
mature-size PsbW is not evident on this gel because it does not stain
with Coomassie Blue. Nevertheless, the appearance of the degradation product can be monitored by immunoblotting (lower panel) and
this smaller protein was purified as shown in Fig. 5B. This
procedure generates near mature-size PsbW that is purified to
homogeneity as judged by silver-staining of polyacrylamide gels (data
not shown) and the protein is hereafter referred to as "mature
PsbW" for simplicity.

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Fig. 5.
Generation of mature-size PsbW by trypsin
digestion. A, pre-PsbW was incubated with immobilized
trypsin for times indicated above the lanes (in min), and the fractions
were analyzed by Coomassie staining of polyacrylamide gels (top
panel) or immunoblotting with PsbW-specific antibodies
(bottom panel). Degradation product evident in immunoblot is
denoted PsbW. B, a sample of pre-PsbW was digested with
trypsin for 3 h (pre-column lane) and then
chromatographed on a mono-Q column. Fractions 1-5 represent column
washes (see "Experimental Procedures") after which the protein was
eluted with a 0-500 mM NaCl gradient (fractions 6-24).
Elution of PsbW was monitored using immunoblotting.
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Fig. 6 compares the CD spectra of
pre-PsbW and mature PsbW in SDS micelles, with both proteins present at
10 µM concentration. The data show that the mature
protein also adopts an
-helical conformation, and this almost
certainly represents the formation of the predicted transmembrane
-helix in the mature protein. Importantly, the mature protein
contains significantly less
-helical structure than the precursor
protein, and we conclude from this finding that the signal peptide in
the pre-PsbW presequence forms an additional
-helix in hydrophobic
environments. These data were quantitated using the cdsstr program (20)
and are shown in Fig. 7 together with a
diagram of the basic structure of pre-PsbW. The CD data predict a total
of 44
-helical residues within the precursor protein and 18 in the
mature protein, which is in reasonable agreement with the overall
structures of the two proteins (Fig. 7B). The two
hydrophobic regions within pre-PsbW account for ~44 residues (of a
137 total), of which 24 are present within the mature protein (17) and
about 20 in the signal peptide. Further analysis is required to
characterize the structure of the loop intermediate in authentic
membranes, but we propose from these data that the precursor
protein does indeed form a two-helix structure in nonpolar environments
and therefore forms a helical hairpin following insertion into the
thylakoid membrane.

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Fig. 6.
Comparison of the CD spectra of pre-PsbW and
trypsin-generated mature PsbW. Pre-PsbW was prepared as shown in
Figs. 2 and 3, and mature PsbW as in Fig. 5, and CD spectra were
recorded in 0.5% SDS. Pre-PsbW, dotted line; PsbW,
solid line.
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Fig. 7.
Secondary structure content of pre-PsbW and
PsbW in SDS micelles. A, the data from Fig. 6 were
quantified using the cdsstr program (20) and are shown expressed in
terms of residues per protein molecule. B, schematic
illustration of the structure of pre-PsbW. The bipartite presequence
contains an N-terminal envelope transit sequence that is removed by
stromal processing peptidase (SPP) in vivo
(although insertion into thylakoids proceeds normally if this domain is
present), followed by a hydrophobic signal peptide. Removal of the
signal peptide is carried out by thylakoidal processing peptidase
(TPP) on the trans side of the thylakoid membrane
after the formation of a loop intermediate. The signal peptide is
capable of forming a 20-residue transmembrane helix according to the
TopPred 2 program (28), and 24 hydrophobic residues are present in the
transmembrane span of the mature protein.
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DISCUSSION |
The early events surrounding post-translational membrane protein
biogenesis have remained unclear in the vast majority of cases.
In vivo, proteins such as pre-PsbW are exposed to two
distinct aqueous environments en route to the thylakoid
membrane: the cytoplasm (prior to chloroplast import) and the stroma
after import. As yet, we do not understand how they avoid aggregation
or whether, for example, chaperone molecules help to maintain the
proteins in an insertion-competent form. The primary technical problems are that membrane proteins tend to require stabilization with detergents after extraction from membranes, and the in vitro
assays used to study protein insertion pathways do not provide any
information on the conformation of the membrane proteins at any stage
prior to membrane insertion. We have therefore sought to prepare
chemical amounts of pre-PsbW to initiate studies into this area.
Of particular interest is the question of when and where the
-helices form in this type of membrane protein. The later stages of
the pre-PsbW insertion pathway involve the formation of a loop intermediate that spans the thylakoid membrane twice with the N and C
termini exposed to the cis face of the membrane and the N
terminus of the mature protein in the lumen (16). The hydrophobic regions in the mature protein and signal peptide are predicted to form
-helices in this loop structure but this has not been tested
experimentally, and we have no information on the timing or location of
-helix formation. Theoretical studies in this area have concluded
that this process must occur before full insertion into the bilayer,
and White and Wimley (23) have proposed that this type of protein could
follow one of two basic insertion pathways: a "water path" in which
the protein folds (i.e. forms
-helices) in the aqueous
phase prior to insertion as a folded entity, or an "interface path"
where the unfolded protein binds to the membrane interface where
-helix formation is promoted and insertion ensues. However, analyses
of the water path are precluded in most cases because of the tendency
of membrane proteins to aggregate in aqueous buffer. Apocytochrome
c has been a popular model system because, despite being a
hydrophilic protein, it has the unusual ability to insert spontaneously
into membrane bilayers. Recent studies have shown that the protein is
largely unstructured in aqueous solution but acquires a highly
-helical structure upon interaction with certain types of lipid,
prior to full insertion (24). Apocytochrome c would thus
appear to be a prime example of an interface path protein. Otherwise,
the best studied cases are of membrane-interactive peptides such as
classical signal peptides (25), relatively hydrophilic mitochondrial
targeting signals (26), and melittin (27). In each of these cases, the
peptide sequence exhibits a relatively extended conformation in
solution but forms an
-helical structure in hydrophobic environments.
Pre-PsbW is by contrast a bona fide membrane protein and
this study has provided information on its structure in both
membrane-mimicking environments (SDS micelles) and aqueous solution.
The CD spectrum of the trypsin-generated mature protein in SDS
indicates that, as predicted from the sequence (17), this protein forms
a transmembrane
-helix in hydrophobic environments. The precursor
protein contains an additional, hydrophobic signal peptide, and such
peptides in other systems have been shown to form
-helices in
membranes (26). Because pre-PsbW contains substantially greater
-helix in SDS than does the mature protein, we propose that its
signal peptide does indeed form an
-helix and that the precursor
therefore forms a helical hairpin once inserted into the membrane.
Pre-PsbW is also a rare example of a membrane protein that is amenable
to analysis in aqueous environments, and on the basis of the data from
this study, we propose that this protein is, like apocytochrome
c, essentially devoid of
-helical structure in the
aqueous phase. The CD structure of the purified Tris-soluble pre-PsbW
shows no detectable
-helix content, and the rapid appearance of this
structure upon addition of SDS indicates that the hydrophobic regions
are available to form
-helical structures in appropriate (i.e. more hydrophobic) environments. However, it is notable
that the tryptophan fluorescence emission spectrum is not consistent with full exposure to solvent, raising the possibility that the protein
may adopt a particular folding state(s) that helps to protect the
hydrophobic regions. One distinct possibility is that the protein may
adopt some form of non-helical hairpin structure in which the two
hydrophobic regions interact with each other. One possibility is a loop
structure that acquires helical structure once in an appropriate
hydrophobic environment.
Although the emphasis in this study has been on the characteristics of
the Tris-soluble monomer/dimer form of pre-PsbW, we should point out
that the data do not rule out the possibility that the initial, 180-kDa
multimeric form is physiologically relevant, because this assembly is
also highly stable in Tris buffer. However, several factors suggest
that this assembly is unlikely to form during the biogenesis of PsbW.
First, proteins are imported into chloroplasts in a largely unfolded
state, and monomeric pre-PsbW is therefore almost certainly released
into the stroma. Second, pre-PsbW is transported into the thylakoid
membrane very rapidly during in vitro assays, and it is
unlikely that this complex form could be assembled under these
conditions because (i) the targeting process is so rapid and (ii)
cell-free translation systems generate very small quantities of
protein, and hence relatively few molecules of pre-PsbW are imported
into a given chloroplast.
Nevertheless, further studies are required to resolve these points, and
it must be pointed out that it also remains to be confirmed whether the
final preparation of purified protein is monomeric or not. Further
studies are also required to determine whether the Tris-soluble protein
is competent for insertion into thylakoid membranes, and insertion
studies with such preparations will certainly help form a coherent
picture of the biogenesis of this class of protein.