(Received for publication, July 26, 1996, and in revised form, September 9, 1996)
From the Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205
OxlT, the oxalate/formate exchange transporter of
Oxalobacter formigenes, was purified as a histidine-tagged
variant, OxlTHis, using Ni2+-linked affinity
chromatography. OxlTHis was readily obtained in high purity (95%)
and reasonable yield (
60%), and showed kinetic and biochemical
features characteristic of its parent, OxlT, including an unusually
high maximal velocity (60 µmol/min per mg of protein at 4 °C).
Circular dichroism spectroscopy of purified OxlTHis identified the
-helix as its dominant secondary structural unit, encompassing
60-70% of OxlTHis residues and consistent with a model suggesting
60% of OxlT (OxlTHis) residues are involved in the construction of 12 transmembrane
-helices (Abe, K., Ruan, Z.-S., and Maloney, P. C. (1996) J. Biol. Chem. 271, 6789-6793). In either
octyl glucoside/lipid or dodecylmaltoside/lipid micelles, solubilized OxlTHis showed a striking substrate-induced
stabilization of function, and at saturating levels of substrate
(1000 × KD) activity recoverable by
reconstitution disappeared with a half-life of 7 days at 23 °C.
Measurement of changes of ellipticity at 222 nm as a function of time
and substrate concentration showed that maintenance of function was
attributable to a substrate-induced stabilization of the
-helical
ensemble with a KD of 10 µM for the
1:1 binding of oxalate to OxlTHis.
The anion transporter, OxlT, carries out the exchange of divalent
oxalate with monovalent formate at the plasma membrane of the anaerobe,
Oxalobacter formigenes (1, 2), and in doing so catalyzes the
vectorial portion of a proton-motive metabolic cycle (3) that drives
ATP synthesis in this organism (1, 3). While OxlT is of interest for
this novel position in cell biology, it also attracts attention for
certain of its biochemical features. For example, studies of OxlT
purified from O. formigenes suggest this carrier operates
with the highest velocity known among transporters of organic molecules
(4). Moreover, examination of crude extracts of the solubilized protein
suggests that substrate binding energy contributes substantially (3.5
kcal/mol) to stabilization of the OxlT-substrate complex (5). As well
as showing the soluble protein retains information relevant to
substrate binding, this last observation suggests that structural
transformation(s) accompanying substrate binding and transport might be
investigated by suitable in vitro experiments with
solubilized protein.
From analysis of the OxlT amino acid sequence (6) one infers a broad
architectural similarity between this protein and other transporters,
in both prokaryotes and eukaryotes, that display the reactions of
antiport, uniport, or symport (3, 7, 8). This large collection of
membrane proteins, now numbering in the several hundreds (3, 4), is
presently exemplified by a few well studied cases, such as the
erythrocyte Cl/HCO3 exchange carrier, Band 3 (9), the red
cell glucose facilitator, GLUT1 (10), and the Escherichia
coli H+/lactose symporter, LacY (11, 12). In these
three examples, biophysical tests indicate the -helix as the main
secondary structural element (9, 13, 14), and this direct experimental
result supports inferences made from more widely available (but
indirect) biochemical and genetic studies that point to the presence of 10-12 transmembrane segments in most examples in this group (3, 7, 8,
13, 15, 29).
Indeed, the transmembrane -helix is often assumed as the dominant
structural element in transporters of this type (16, 17), but except
for the cases noted this supposition has not been tested directly. For
this reason, and because of the unusual kinetic features of OxlT, the
initial objective of work reported here was to assess secondary
structure in this transporter. To do this, we installed a histidine tag
at the OxlT C terminus and purified the tagged protein, OxlTHis, by
affinity chromatography using a Ni2+-linked adsorbent. The
CD spectrum of solubilized, purified OxlTHis shows that the majority of
its residues are organized in an
-helical configuration, supporting
a generic model in which this and similar transporters contain 10-12
transmembrane
-helices. Our second objective was to use the purified
protein to correlate structure and function. Such studies show that the
loss of activity observed in the absence substrate reflects a
spontaneous breakdown of the
-helical ensemble.
pBKOxlT was constructed by insertion into pBluescript II SK+ (Ampr) of a 1.4-kilobase XbaI-HindIII fragment encoding OxlT (6). In this construct, the first of two in-frame OxlT UAA stop codons are flanked by BsrGI and NheI sites. To make an expression vector encoding histidine-tagged OxlT, we digested pBKOxlT with both BsrGI and NheI and then ligated the products with a synthetic BsrGI-NheI bridging oligonucleotide specifying nine consecutive histidine that would extend from the OxlT C-terminal histidine. The sequence of the resulting vector, pBDOxlTHis, was confirmed by sequencing of double stranded DNA using the dideoxy chain termination method of Sanger (18). pBKOxlT and pBDOxlTHis were expressed in XL3, a strain that also carries pMS421 (specr, LacIq) to ensure repression of OxlT and OxlTHis in the absence of induction (6).
Expression of OxlT and OxlTHisA single colony of XL3
carrying either pBKOxlT or pBDOxlTHis was dispersed in 5 ml of LB broth
with antibiotics (100 µg/ml ampicillin, 50 µg/ml spectinomycin);
after overnight growth with vigorous shaking, cells were added to 0.5 liter of fresh media and grown until A600
reached 0.2-0.3, at which point 1 mM
isopropyl-1-thio--D-galactopyranoside was added to
induce expression of OxlT or OxlTHis. Four hours later, cells were
harvested by a 10-min centrifugation at 4,000 × g and
lysed by incubation at 37 °C for 15 min in 50 ml of lysis solution
(300 µg/ml lysozyme, 40 µg/ml DNase, 0.5 mM freshly
dissolved phenylmethylsulfonyl fluoride) (19). The resulting ghosts
were washed twice with iced distilled water, and membrane proteins were
extracted at pH 7 by incubation for 60 min on ice with 25 ml of
solubilization buffer (20 mM potassium phosphate, 6 mM
-mercaptoethanol, 20% glycerol, 0.42% acetone/ether
purified E. coli phospholipid, 1.5% octyl glucoside)
containing 10 mM potassium oxalate. The mixture was cleared
of cell debris and unextractable material by a 30-min centrifugation at
145,000 × g; the remaining crude extract, usually
about 300 µg of protein/ml, was stored at
80 °C.
To purify OxlTHis, a 5-ml Quik-Sep column (Isolab) was packed with 0.2 ml of Ni2+-NTA resin (Qiagen), and the resin was washed at 4 °C with 20 ml of distilled water. The column outlet was sealed with Parafilm, 3 ml of crude detergent extract was added, and after sealing the column inlet, the resin and extract were mixed by gentle rotation for 4 h at 4 °C. The extract, depleted of OxlTHis, was allowed to drain from the column, and nonspecifically bound residual material was removed by a 20-ml wash at 1 ml/min, using solubilization buffer supplemented with 200 mM sodium fluoride and 50 mM imidazole, along with OxlTHis substrates (10 mM potassium oxalate or 100 mM potassium formate) as necessary. OxlTHis was eluted by centrifugation after a 20-40 min incubation with 0.2 ml of solubilization solution containing either 500 mM imidazole or 200 mM EDTA, each neutralized by potassium hydroxide, with or without OxlT substrates.
Reconstitution of OxlTHis and OxlTTo compare the transport activities of OxlTHis and OxlT, we first set the two proteins at approximately equal concentrations by mixing 1 volume of purified OxlTHis or a crude cell extract containing OxlT with 149 or 9 volumes of solubilization buffer, respectively. Reconstitution from these mixtures then followed earlier procedures (1, 4, 20). Briefly, the proteins were dispersed in lipid/detergent micelles by a sequential mixing of the following components: 14.6 µl of 100 mM potassium phosphate (pH 7.0); 4.5 µl of 15% octyl glucoside; 35 µl of 45 mg/ml bath-sonicated E. coli phospholipid; and 200 µl of the OxlTHis or OxlT mixtures. The suspensions were kept on ice for 20 min, after which oxalate-loaded proteoliposomes were formed by adding 5 ml of 23 °C loading buffer (100 mM potassium oxalate, 50 mM potassium phosphate, pH 7) to bring the detergent level below its critical micellar concentration. After an additional 20 min, proteoliposomes were used to determine OxlTHis or OxlT transport activity in either of the two assays outlined below.
Assays of Oxalate TransportIn routine work, oxalate transport was determined at 23 °C by a simplified assay (4) in which replicate 0.1-ml portions of the proteoliposome suspension were applied to the center of a pre-soaked Millipore GS filters (0.22 µm pore size). After removing external loading buffer by a wash with 5 ml of assay buffer (100 mM potassium sulfate, 50 mM potassium phosphate, pH 7), the vacuum was interrupted, and the filter with entrapped proteoliposomes was overlaid with 0.3 ml of assay buffer containing 0.1 mM [14C]oxalate. The oxalate exchange reaction was complete by 4 min, at which time the assay was terminated by washing the filter twice with 5 ml of assay buffer. For this fixed-time assay, activity is reported as micromoles of [14C]oxalate incorporated per mg of protein during the 4-min incubation.
To measure the kinetic parameters of oxalate transport, proteoliposomes were isolated by centrifugation (20), washed once with assay buffer, and after resuspension in assay buffer, the time course of oxalate transport was measured at 4 °C by incubation with [14C]oxalate of the indicated concentrations. Reactions were terminated by filtration and washing (1, 18).
Electrophoresis and Western BlottingSamples of fractions obtained during purification of OxlTHis were subjected to SDS-PAGE1 using 12% acrylamide, as described (21), and protein content was evaluated by staining with Coomassie Brilliant Blue. The position of OxlTHis was verified by Western blot of a duplicate gel in which proteins were transferred to nitrocellulose (22) and exposed to a polyclonal antiserum selective for the OxlT N terminus (6). Binding of the primary antibody was visualized by chemiluminescence (Amersham) (6).
CD SpectroscopyCD spectra were measured for OxlTHis which had been passed over a Centri-Spin20 desalting column (Princeton Separation) to remove the EDTA used for elution. Unless noted otherwise, the column was prehydrated with 0.5 ml of solubilization buffer (with or without added substrate, according to the experimental design), and residual buffer was removed by a 2-min centrifugation at 750 × g. EDTA-eluted OxlTHis (80 µl) was placed on the column, and the desalted product was collected by a second centrifugation; as a blank, a solution containing all components except OxlTHis was prepared in parallel.
CD spectra were collected using an AVIV 60DS circular dichroism
spectropolarimeter; calibration with (+)-10-camphorsulfonic acid gave a
ratio of 2.25 for ellipticities at 192.5 and 290.5 nm. The CD
spectrum of OxlTHis (0.2-0.5 mg/ml in protein) was measured in a
0.01-cm path length quartz cell, with temperature set to 25 °C
unless otherwise noted. Data were obtained at constant slit width (1.5 nm), and each spectrum was acquired as the result of five repeated
scans from 260 to 190 nm, using scan intervals of 0.5 nm and an
integration time of 1 s at each wavelength. The time for data
collection was about 20 min for each set of five scans. Blank and
experimental samples showed identical responses above 250 nm,
corresponding to +1 millidegree, suggesting the absence of significant
differential scattering of left or right circularly polarized light by
detergent/lipid micelles (23); blank values remained at +1 millidegree
as the wavelength was reduced to about 215 nm and then rose to +3
millidegrees by 190-195 nm. Experimental samples showed peak negative
and positive deflections from the blank at 220-222 nm and 190-195 nm,
respectively; depending on protein concentration, at these wavelengths
experimental samples showed uncorrected signals of between
4.5 to
12 millidegrees (220-222 nm) and +20 to +50 millidegrees (190-195
nm), indicating adequate signal-to-noise ratios within the range
investigated. Raw spectra were corrected by subtraction of baseline
spectra obtained using the blank solution, and after smoothing, the net spectra were used for estimation of OxlTHis
-helical content.
CD spectral measurements were expressed as mean residue ellipticity, in
units of degree cm2 dmol1, according to: mean
residue ellipticity = (100 ·
· M)/(c · d · n), where
is measured ellipticity (deg), M is molecular weight, c is protein concentration (mg/ml), d is path length (cm), and
n is the number of residues in OxlTHis (n = 427). Spectral decomposition was done by least-squares fit
(Sigma plot) of mean residue ellipticity values at
200-260 nm to the contributions of four components (
-helix,
-strand,
-turn, and unordered) taken from the basis set tabulated
by Yang et al. (24) or Park et al. (25),
constraining the sum of contributions to equal 1. Because measurements
at wavelengths below 200 nm were not considered, only estimates of
-helix content are reported (26). For the basis set of Yang et
al. (24), an average helix length of 24 residues was used to
account for the dependence of ellipticity on helix length (24).
Protein was estimated by a modification of the method of Schaffner and Weissman (27).
To enable convenient purification of OxlT, we exploited Ni2+-linked affinity chromatography. The principle underlying this approach is that a sequence of six or more tandem histidine residues can form a relatively tight binding pocket for the divalent nickel cation, allowing a suitably tagged protein to be retained by a Ni2+-containing resin (28). Accordingly, we prepared OxlT variants having 10 consecutive histidine residues at either the N terminus (HisOxlT, not described) or the C terminus (OxlTHis, see "Experimental Procedures"). Because preliminary trials (not given) indicated that the latter showed somewhat tighter binding to the Ni2+-linked resin, we focused on purification and characterization of OxlT having the C-terminal histidine tag.
To purify OxlTHis, we first prepared membrane ghosts by lysis of cells
overexpressing this protein and then solubilized membranes using octyl
glucoside in the presence of excess E. coli phospholipid, with glycerol added as the osmolyte stabilant (20). As anticipated (4,
6), the SDS-PAGE profile of the detergent extract showed OxlTHis as a
protein with an apparent mass near 35-kDa (Fig. 1, left). OxlTHis was depleted from the extract by incubation
with the Ni2+-linked adsorbent, and reappeared only when
wash conditions were altered so as to release Ni2+-linked
materials (Fig. 1, left). Western blot analysis verified these findings. Thus, only the crude extract and EDTA-eluate contained material reactive with an antibody directed to the OxlT N terminus (Fig. 1, right). In each case, the major element moved as a
35-kDa protein, corresponding to monomeric OxlTHis; dimeric and
trimeric OxlTHis (4, 6) were evident in the Western blot (about 75 and
125 kDa, respectively), but were not seen in the Coomassie-stained gel
due to their low abundance.2 Since proteins
other than OxlTHis were not detected (Fig. 1, left), we
concluded that OxlTHis is readily prepared at high purity (95%) by
Ni2+-linked affinity chromatography.
The function of purified OxlTHis was assessed by [14C]oxalate transport into oxalate-loaded proteoliposomes, and in the abbreviated assay used to monitor the purification, EDTA-eluted OxlTHis showed an activity of 92 µmol/mg of protein (Table I); material of equivalent activity (114 µmol/mg of protein) was found in experiments that used 500 mM imidazole for elution (Table I). Because these values compare favorably with that observed after purification of OxlT by conventional methods from O. formigenes (60 µmol/mg of protein) (4), we conclude that heterologous expression of OxlT (OxlTHis) in E. coli has no effect on the intrinsic properties of this transporter.
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For the experiment illustrated by Fig. 1, densitometry of the Coomassie-stained gel indicated OxlTHis comprised 8.4% of the initial crude detergent extract. Since purified OxlTHis was recovered at about 10-20 µg of protein/ml crude extract, or about 5% of the starting material (4.8 ± 1.2% (mean ± S.E.) in six experiments), overall recovery was near 60% (new work indicates that yield may be increased by extending the time of exposure to EDTA during elution). Given OxlTHis as 5-10% of the crude extract, one expects a 10-20-fold increased specific activity if the purified protein is fully functional. Since the mean specific activity of the pure product was 15-19-fold increased over the crude extract (Table I), it seemed likely that OxlTHis was obtained with little if any loss of function.
In additional work, we conducted a more detailed comparison of the
transport activities of purified OxlTHis and its parent, OxlT; to
obtain quantitative information, kinetic behavior was monitored at
4 °C, since OxlT-mediated transport is unusually rapid (maximal
velocity of about 1 mmol/min per mg of protein at 23 °C) (4). Such
tests established that for the usual conditions of assay the time
course of oxalate exchange was the same for OxlTHis and OxlT
(half-times of about 5 min) and that, as expected for simple antiport,
the reaction could be described as an exponential approach to
equilibrium (Fig. 2A). The two proteins also
had similar Michaelis constants for [14C]oxalate
transport (135 and 145 µM, respectively, for OxlTHis and
OxlT) (Fig. 2B), although the maximal velocities of the two preparations differed considerably (60 versus 4.3 µmol/min
per mg of protein, respectively). This last finding was expected, since
OxlTHis function had been assessed with purified material, whereas OxlT
activity was measured in a crude extract (e.g. Table I).
These observations, together with the information noted above, lead us
to conclude that the histidine-tagged OxlTHis retains the essential
aspects of catalytic activity of its parent, OxlT.
Stability of OxlTHis
OxlTHis had been purified to
evaluate its secondary structure by CD spectroscopy, and because this
would require extended incubation of solubilized protein at 25 °C
(or higher), it was essential to document the stability of OxlTHis for
these general conditions. For this reason, solubilized (and desalted)
OxlTHis was dispersed in mixed lipid-detergent micelles in the presence of 10 mM oxalate (to take advantage of
substrate-stabilization (5)), and at intervals during incubation at
room temperature samples were withdrawn to measure residual activity by
reconstitution. The analysis was done with both octyl glucoside and
dodecylmaltoside as solubilizing agents, since these are the detergents
most commonly used for solubilization of bacterial membrane proteins.
For each of these preparations, 50-60% of the initial activity was
recovered after 7 days incubation (Fig. 3), indicating
that purified OxlTHis was sufficiently stable for spectral
analysis.
CD Spectroscopy of OxlTHis
Early work showed that solubilized OxlT retains a capacity to bind its substrates (5), implying that the solubilized material has structural information relevant to the overall process of membrane transport. Consequently, it seemed reasonable to use CD spectroscopy to probe secondary structure of the solubilized protein.
The CD spectra recorded for solubilized OxlTHis (Fig.
4, panel 2) showed features expected for a
protein with a predominantly helical structure, that is, negative
deflections in mean residue ellipticity above about 200 nm, with a
minimum near 222 nm, and stronger positive deflections below 200 nm,
with a maximum near 195 nm. Accordingly, one may conclude that OxlTHis
(and OxlT) contains the -helix as its main structural element. This
overall structure was not influenced by the presence of bulk lipid,
since an equivalent spectrum was recorded for protein eluted from
Ni2+-agarose and further processed with lipid-free buffer
(Fig. 4, panel 3). It was also evident that the spectra of
OxlTHis bound to oxalate or formate were nearly superimposable (Fig. 4,
panel 2), while the spectrum taken in the absence of
substrate (Fig. 4, panel 4) had both its minimum (near 222 nm) and maximum (near 195 nm) reduced in absolute value. Since there
had been gradual decline of these peak values during the repeated scans
required for signal averaging (not shown), it appeared that the helical nature of OxlTHis became progressively less prominent with continued incubation in the absence of substrate. This behavior was not studied
further in these experiments (but see below), except to note that it
correlated with loss of function, as shown by a separate experiment in
which activity was monitored subsequent to the recording of CD spectra.
In those cases, there was essentially complete recovery of
[14C]oxalate transport activity for samples incubated
with oxalate or formate (95 or 80% recovery, respectively), but only a
30% recovery for the sample processed without substrate.
In the second part of the experiment shown by Fig. 4, each sample was exposed to elevated temperature (55 °C) before recording its CD spectrum in a single 3-min scan. Elevated temperature had little apparent effect on samples in which substrate was present, even after a 50-55 min incubation (Fig. 4, panels 6 and 7), but in the absence of substrate, the signal became so severely degraded that heating was discontinued after only 15 min (Fig. 4, panel 8). This maintenance of an organized helical structure at high temperature reinforces the idea that the OxlTHis-substrate complex has an unusual stability (Ref. 5, and see below).
CD spectra collected in this (Fig. 4) and two other experiments (not
shown) were used for estimation of secondary structure using basis
spectra describing the responses of the -helix,
-sheet,
-turn,
and random coil configurations, constraining the sum of the fractional
contributions to equal 1. To reconstruct experimental spectra, a
least-squares analysis was performed using the basis set of Yang
et al. (24) (Fig. 4, panel 1) or Park et
al. (25) (Fig. 4, panel 5), both of which derive from
analysis of globular proteins of defined structure. The OxlTHis spectra
were not well accommodated over their entire span (190-260 nm) by
either basis set, largely because of deviations at wavelengths below
200 nm (not shown). Since the background signal increased significantly in this region, poor fit was attributed in part to non-protein chiral
signals that were not removed by background subtraction. To improve
overall fit, therefore, the experimental spectra were truncated at 200 nm, and with this modification it became possible to obtain consistent
findings with regard to the
-helical composition of OxlTHis. When
calculations were based on the reference set of Yang et al.
(24), OxlTHis was best described as containing 62-67%
-helix in
the presence of substrates and 52%
-helix in their absence (at
25 °C) (Table II, Method I), whereas the Park et al. (25) basis set gave helix contents as 72-74% (with
substrates) or 65% (without substrate) (Table II, Method II).
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While each method of calculation gave a similar estimate of OxlTHis
helical content (Table II), the calculated contributions by -sheet,
-turn, and unordered regions varied significantly (not shown). This
had been anticipated, since different assumptions are embodied by the
different basis sets (e.g., compare Fig. 4, panels
1 and 5), and because estimates of non-helical
structures are inaccurate unless the experimental spectra extend to 184 nm or below (26). As a result, while it was not possible to assess the
contributions of
-sheet,
-turn, or random coil, there was consistent evidence that the
-helix comprises the dominant
structural element, encompassing about 60-70% of OxlTHis
residues.
Prior studies had
noted a relatively rapid loss of OxlT activity (half-life of 6-7 min
at 37 °C) when crude extracts were not supplemented with substrate
(5). An explanation for this phenomenon was suggested by the present
observations that incubation in the absence of substrate was associated
with loss of both function and helical structure (Fig. 4 and text).
Thus, it was feasible that substrate binding to OxlTHis might act to
stabilize the helical ensemble itself. To test this idea, we measured
the time course of changes in ellipticity at 222 nm as a function of
oxalate concentration, using temperature (37 °C) and substrate
levels (0-1000 µM) that gave measurable changes in
ellipticity over a time interval (5 min) minimizing baseline drift.
This work (Fig. 5) confirmed the strong relationship
between substrate binding and preservation of helical structure. If one
assumes the binding of oxalate and OxlTHis is associated with a change
in protein structure from a conformation that decays spontaneously
(half-life of 4-5 min at 37 °C) to one that is stable on the time
scale relevant to the experiment (e.g. Fig. 3), these data
are accommodated by the 1:1 binding of oxalate and OxlTHis with a
dissociation constant (KD) of 10 µM
(Fig. 5, inset). Such findings establish a direct
correlation between the function of OxlTHis and its helical content.
The OxlT amino
acid sequence (6) indicates it has a global structure resembling any
one of a large number of transport systems that mediate the reactions
of symport, uniport, or antiport (3). The transmembrane -helix is
usually taken as the main structural element for these and many other
transporters, a presumption that is rarely supported by experiment. For
this reason, and because early work with crude extracts had shown OxlT
to have unusual stability as a solubilized protein (5), we reasoned
that direct and useful information could be gained by CD spectroscopy
of OxlT if a suitably purified preparation were available.
OxlT had been purified earlier using traditional techniques (4), but as is often the case with membrane proteins, the tendency of OxlT to associate in non-stoichiometric fashion with detergents and phospholipids leads to a heterogeneity in both size and charge that makes it difficult to apply conventional methods of purification in predictable fashion. It is possible to simplify and accelerate this process by affinity methods (30-32), and we elected to use Ni2+-linked chromatography of a histidine-tagged variant (OxlTHis), a tactic that enabled the convenient production of highly purified material on a routine basis. Purified OxlTHis has the specific activity expected of the fully functional protein (Table I and text), as one might expect from the finding that (with substrate present) OxlTHis lifetime is long compared to the time required for its purification. OxlTHis also retains the kinetic features of its parent, including its unusually high maximal velocity (Fig. 2); this corresponds to a turnover number of about 100/s at 4 °C, since only half the protein used for reconstitution is incorporated by proteoliposomes (4). Clearly, analysis of OxlTHis is relevant to understanding its parent, OxlT.
CD Spectroscopy of OxlTHisIn extracting structural
information from CD spectroscopy, one makes four general assumptions
(24-26, 33): (i) that the CD spectrum arises as the linear combination
of its component spectra (i.e. those of the -helix,
-sheet,
-turn, unordered regions, etc.); (ii) that these
component, or basis, spectra may be deduced from the behavior of
reference proteins whose structures are understood; (iii) that an
appropriate set of reference proteins is available; and (iv) that the
experimental spectrum extends over a sufficiently wide range to provide
statistically significant information about the component spectra. Of
these assumptions, the last two are at issue in the work described
here. It is acknowledged, for example, that basis spectra deduced for
the
-pleated sheet,
-turn, and random coil configurations are
sensitive to the choice of the reference set (33) (e.g. Fig.
4, panels 1 and 5), and an inability to obtain
accurate spectral information below 195-200 nm (as in these
experiments) further limits one's confidence in attempts to evaluate
such components (26). These are the likely explanations for failure to
derive consistent estimates for these structures in OxlTHis, as well as
for the relatively poor fit by theoretical reconstructions when helix
content was significantly diminished (e.g. Fig. 4,
panel 8). By contrast, it is generally held that the
-helix yields a signal of relative uniformity in different environments (but see below), and since the helix chiral signal dominates in the more readily detected wavelengths, estimates of this
parameter are the more likely to be accurate in the range considered
here (
200 nm), especially if the target has a substantial helical
content (26, 33). In fact, the CD spectrum of solubilized OxlTHis has
the profile expected of a largely helical protein (Fig. 4), and
calculations using two different basis sets gave similar estimates of
OxlTHis helical content (Table II). One might also note that as
structural information on membrane proteins is limited, analysis of the
OxlTHis CD spectrum relies on component spectra derived from globular
proteins, and this may carry potential risk. As an example, it has been
proposed that the transmembrane
-helix differs from the
-helix
exposed to water in having a somewhat stronger signal than expected
(33), and this idea, still under discussion, raises the possibility
that standard calculations (e.g. Table II) may overestimate
the helix content of membrane proteins. Nevertheless, for the
photosynthetic reaction centers of Rhodobacter viridis and
Rhodopseudomonas sphaeroides, whose structures are known
from crystallography, such error appeared to be small, and Method I
(Table II) overestimated helix content by only about 10% (39%
observed versus 42.5% calculated and 51% observed
versus 56% calculated, respectively) (33). Similarly, Method I provided a helical content for the nicotinic acetylcholine receptor (23%) (34) consistent with electron diffraction data (35).
It appears probable that the structure of solubilized OxlTHis resembles
that adopted by this protein in the lipid bilayer. Although this
assumption was not tested here in any specific way, it has been
verified for the acetylcholine receptor (34) and the water channel,
CHIP28 (36). And for the LacY symporter, presumed to resemble OxlT in
general structure, overall helix content was not different for the
solubilized and reconstituted protein (cited in Ref. 13). In addition,
since tests with crude preparations of OxlT indicate the solubilized
protein binds its substrates (5), we take the information reported here
as relevant to understanding structure-function relations in both
solubilized and membrane-bound OxlT (OxlTHis). Accordingly, given a
helical content of 60-70% (Table II), one expects that 256-295 of
the 427 OxlTHis residues are organized in this way, suggesting that OxTHis could have 10-13 -helices of sufficient length (23-25 residues) to span the lipid bilayer. The OxlTHis CD spectrum is therefore consistent with models derived from hydropathy analysis, which predict 11 or 12 transmembrane
-helices (6). Inasmuch as
hydropathy analysis and other indirect methods generate similar models
for a large number of transporters (3, 7, 11), our findings, together
with those of the LacY symporter (13), the band 3 anion exchanger (9),
and the glucose carrier (14) reinforce this as a generic feature of
such transporting systems.
More important than an overall consistency between such models and experiment, this work now establishes a quantifiable relationship between structure and function for OxlTHis. This was first noted in a qualitative way by the parallel reduction of both activity and helical content during spectral analysis (Fig. 4). Subsequently, we explored this explicitly by measuring the kinetics of spontaneous decay of helix content and by showing that a KD for oxalate could be derived from the substrate dependence of this process (Fig. 5). We had earlier used reconstitution of activity as the method of analysis, and while that gave a similar estimate of KD (20 µM [5] versus the 10 µM recorded here), such observations remained a phenomenological description without clue as to underlying mechanism. The present work confirms the initial findings (5) and suggests as well that loss of function in the absence of substrate arises from a breakdown in helical structure. Such observations offer strong evidence that the solubilized protein binds substrate, likely in a 1:1 stoichiometry (Fig. 5, legend), and show how CD or other spectroscopic tools might assess secondary structure for the solubilized transporter.
We note in conclusion that purified OxlTHis can be maintained for an extended period in detergent/lipid micelles without loss of function, provided that substrate is present (Fig. 3). Since it has been difficult to study membrane transporters after their extraction from the lipid bilayer, our work establishes OxlT as a robust model with which to investigate correlations between structure and function using the solubilized material.
We acknowledge fruitful discussions with Drs. Sriram Subramaniam and David Shortle of the Johns Hopkins University Medical School and Dr. Reinhart Reithmeier of the University of Toronto. We also thank Dr. Gerald Fasman of Brandeis University for providing us with the spectral basis set developed by Park et al. (25). We are especially grateful to Dr. Keietsu Abe and Joel Gillespe for their advice on technical aspects of this work.