JOURNAL OF BACTERIOLOGY, Feb. 2006, p. 894–901 Vol. 188, No.

3
0021-9193/06/$08.00⫹0 doi:10.1128/JB.188.3.894–901.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Topology and Boundaries of the Aerotaxis Receptor Aer in the

Membrane of Escherichia coli
Divya N. Amin, Barry L. Taylor, and Mark S. Johnson*
Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, California 92350
Received 11 September 2005/Accepted 10 November 2005

Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a
cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that

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both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic
segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a
single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating
that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the
overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The
proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and
extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and
to decrease lateral diffusion in the membrane, at 4°C. Membrane boundaries were identified in membrane
vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic
residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4ⴕ-maleimidylstil-
bene-2, 2ⴕ disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with
two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor
contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid
and peptide replacements.

Escherichia coli has five chemoreceptors that guide cells to ment tend to be cytoplasmic, whereas negatively charged res-
favorable environments (41, 47). Four of these are methyl- idues near boundaries of a TM segment tend to be exported
accepting chemoreceptors (MCPs) that bind periplasmic li- (10, 14, 29, 59). Furthermore, central proline residues in model
gands and transmit this information across the membrane to systems can convert a 40-residue single membrane-spanning
the cytosolic two-component chemotaxis cascade. The fifth hydrophobic polymer into two membrane-spanning helical seg-
receptor, Aer, is an aerotaxis, energy, and redox sensor con- ments (44). However, the central Pro186 present in E. coli Aer
taining N-terminal PAS sensing and C-terminal signaling do- is not conserved in other Aer proteins, so its influence may not
mains separated by a putative membrane anchor (1, 2, 52, 56). be important for membrane topology. From a survey of known
Although not proven, several lines of evidence indicate that membrane protein structures (19), the length of the Aer mem-
both PAS sensor and C-terminal signaling domains of Aer are brane anchor meets the minimum requirements to span the
cytosolic. (i) All known PAS domains are intracellular sensors membrane once, extrude into the periplasm, and return to the
(62, 68). (ii) Native folding of the N-terminal PAS domain cytosol (Fig. 1). However, a number of other conceivable struc-
requires HAMP domain residues that are C terminal to the tures (e.g., parallel to the bilayer surface) could occur if this
membrane anchor (18). (iii) Mutations in the HAMP domain segment were unable to span the membrane twice (54, 55).
are suppressed by mutations in the PAS domain (65). (iv) GFP To determine the overall topology of the Aer homodimeric
fusions to Aer N termini (D. Salcedo and M. S. Johnson, protein, we used a cysteine-scanning approach with whole cells
unpublished data) or C termini (11) fluoresce. Since GFP (33) and with membrane vesicles (3, 23, 35). We estimated the
fluoresces in the cytosol but not in the periplasm (11), both N proximity of cognate cysteine replacements by measuring the
and C termini are likely cytosolic. Thus, a topology similar to rate and extent of dimer formation after the addition of the
that of MCPs, where the sensor is periplasmic and the signaling oxidant copper phenanthroline. Membrane boundaries were
region is cytosolic, is not likely. identified by using a series of sulfhydryl-reactive probes (3, 23,
Aer has just one hydrophobic segment long enough to span 35). We show that the Aer membrane anchor spans the mem-
the membrane. The region exhibits several hallmarks consis- brane twice and contains a central, flexible loop that faces the
tent with two membrane-spanning segments separated by a periplasmic space. However, the orientation between cognate
hairpin loop. These include ⬃38 consecutive hydrophobic res- transmembrane helical faces could not be identified, due to the
idues, a central proline (P186), and flanking N- and C-terminal sparse cross-linking within the membrane core.
arginines (Fig. 1). It is known that successive positively charged
residues near the boundaries of a transmembrane (TM) seg-
MATERIALS AND METHODS
* Corresponding author. Mailing address: Division of Microbiology Reagents. Methoxy polyethylene glycol 5000 (mPEG) was purchased from
and Molecular Genetics, Loma Linda University, Loma Linda, CA Nektar Therapeutics (Huntsville, AL); 4-acetamido-4⬘-maleimidylstilbene-2,2⬘-
92350. Phone: (909) 558-4480. Fax: (909) 558-4035. E-mail: mjohnson disulfonic acid, disodium salt (AMS) was from Molecular Probes, Inc. (Eugene,
@llu.edu. OR); 5-iodoacetamidofluorescein (5-IAF) was from Pierce (Rockford, IL); PE-

894
VOL. 188, 2006 TOPOLOGY AND BOUNDARIES OF Aer IN E. COLI MEMBRANE 895

containing a 2.5 mM final concentration of NEM (32). To limit lateral diffusion

of Aer in the membrane, a parallel reaction was carried out at 4°C with cells and
oxidant that had been precooled separately at 4°C for 10 min prior to initiation
of the reaction. After 20 min at 4°C, the reaction was quenched with stop solution
containing NEM (as above) and incubated for an additional 10 min at 4°C before
being boiled. Samples were run on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under nonreducing conditions and Western blot-
ted. A control was used to test for artifactual cross-linking during the denatur-
ation step by incubating cells with 2.5 mM NEM prior to oxidation with copper
phenanthroline. The percentage of cross-linking was calculated by dividing the
intensity of the cross-linked dimer band by the sum of the intensities of the
monomer and dimer bands, multiplied by 100. BT3312/pGH1 (38) and BT3312/
pMB1 were used as positive and negative cross-linking controls, respectively. The
extent of cross-linking for 10 min at 23°C and 20 min at 4°C was compared for all
cysteine replacements.
Preparation of membrane vesicles. Bacterial membranes containing wild-type

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or mutated Aer receptors expressed at approximately 10% of the total mem-
brane protein were prepared as previously described by Butler and Falke (4) with
several modifications. Five milliliters of overnight cultures was inoculated into
250 ml of H1 minimal salts medium. Cultures were shaken at 30°C, induced with
0.6 mM IPTG at an optical density at 600 nm of 0.4, and grown for an additional
3 h. Cells were harvested by centrifugation at 10,000 ⫻ g for 10 min at 4°C and
resuspended in 4 ml of low-salt buffer (100 mM sodium phosphate [pH 7.0], 10%
glycerol, 10 mM EDTA, 1 mM 1,10-phenanthroline containing freshly added 46
mM dithiothreitol [DTT], and 2 mM PEFAbloc). Cells were disrupted by three
freeze-thaw cycles, lysed by sonication (Branson Sonifier cell disrupter 200) at
60% power (three 15-s bursts with 45-s pauses) in an ice-salt bath, and centri-
fuged at 12,000 ⫻ g for 20 min at 4°C. Membranes were pelleted by centrifuga-
tion at 485,000 ⫻ g for 20 min and washed three times as follows. Membrane
pellets were resuspended by sonication in high-salt buffer 1 (20 mM sodium
phosphate [pH 7.0], 2 M KCl, 10% glycerol, 10 mM EDTA, 1 mM 1,10 phenan-
throline containing freshly added 5 mM DTT, and 2 mM PEFAbloc), pelleted,
resuspended in high-salt buffer 2 (without DTT or 1,10-phenanthroline), pel-
leted, resuspended in final buffer (with no DTT; 1,10-phenanthroline; or KCl),
pelleted, resuspended in 200 ␮l of final buffer, aliquoted, frozen in a dry ice-
FIG. 1. Hypothetical membrane anchor topology for the dimeric ethanol bath, and stored at ⫺80°C. The concentration of protein in the mem-
Aer receptor. (A) A helix-loop-helix membrane anchor would accom- brane was measured by a BCA (bicinchoninic acid) assay (Pierce Chemical)
modate a cytosolic placement for both PAS and signaling domains. using bovine serum albumin as the standard. Frozen membrane preparations
(B) An expanded view of the membrane region (highlighted in gray) were diluted to the required concentration with final buffer prior to use.
for one Aer monomer, including the residues examined in this study. Accessibility studies with 5-IAF. These experiments were modifications of the
protocol described by Boldog and Hazelbauer (3). Membrane vesicles containing
between 5 and 15 ␮g of protein in 12 ␮l of final buffer were incubated with 5-IAF
(500 ␮M in dimethyl formamide) in the presence and absence of 1% SDS to label
FAbloc was from Centerchem (Norwalk, CT); and N-ethyl maleimide (NEM) the denatured and native forms of the protein, respectively. The SDS-treated
was purchased from Sigma (St. Louis, MO). reaction mixture was boiled for 5 min, whereas the native sample was incubated
Membrane prediction algorithms. The membrane-spanning segments of Aer at 23°C for 5 min. Both reactions were stopped by the addition of 10 ␮l of SDS
were predicted by analyzing the entire Aer sequence with the following pro- sample buffer containing 5% ␤-mercaptoethanol. Samples were boiled for 5 min
grams: MEMSAT (25, 27), DAS (8), PHDhtm (57, 58), TopPred (7, 64), and subjected to SDS-PAGE. Immediately after electrophoresis, wet gels were
TMHMM (30), TMpred (21), HMMtop (version 2.0) (63), TMAP (50, 51), analyzed for fluorescein fluorescence with an Alpha Innotech gel documentation
TMfinder (12), PRED-TMR (49), SPLIT 4.0 (28), and SOSUI (20). system with a UV light box. The total protein in each Aer–5-IAF band was
Bacterial strains and plasmids. BT3312 (aer tsr) (56) is a derivative of E. coli estimated by staining the gel with Coomassie blue. The percent accessibility for
strain RP437, which is the wild type for aerotaxis (48). Plasmid pMB1 is a
each residue was calculated by taking the ratio of native to denatured Aer
cysteine-negative (C-less; Aer-C193S/C203A/C253A) derivative of pGH1 (wild-
fluorescence, dividing by the ratio of native to denatured Aer protein, and
type Aer) (52) and was derived by digesting pGH1 with SmaI and SalI and
multiplying by 100.
replacing Aer by a C-less Aer construct previously engineered in pSB20 (2, 38).
Accessibility studies with mPEG. Between 4 and 6 ␮g of membrane vesicles in
Both pGH1 and pMB1 are derivatives of pTrc99A, under the control of the
13 ␮l of final buffer was incubated with mPEG (5 mM final concentration).
isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible ptrc promoter (52).
Parallel reactions of native proteins were carried out at 23°C and at 4°C for 1 h.
Single-cysteine replacements in the membrane anchor region (residues 163 to
The reactivity of the denatured forms was determined in the presence of 1% SDS
210) of Aer were made in pMB1 using the Quik Change site-directed mutagen-
esis kit from Stratagene (La Jolla, Calif.). Each plasmid was transformed by heat at ⬃100°C for 5 min. Reactions were stopped by the addition of 6.75 ␮l of SDS
shock into BT3312. Expression of the Aer protein was confirmed by Western blot sample buffer containing 5% ␤-mercaptoethanol. Samples were boiled for 5 min,
analysis using antisera against Aer2–166 (56), and the mutation was confirmed by run on SDS-PAGE, and Western blotted. Accessibility to mPEG was measured
DNA sequencing. All cysteine replacement constructs were inoculated on semi- by comparing the accessibilities under native and denaturing conditions.
solid succinate swarm plates containing ampicillin (100 ␮g ml⫺1) to assess aero- Preblocking accessible cysteines with AMS in intact cells. Whole intact cells
tactic behavior as described previously (2). with single-cysteine replacements in the membrane anchor were incubated with
In vivo cross-linking using copper phenanthroline. Cells expressing single- or without AMS (8 mM final concentration) in final buffer for 45 min at 23°C.
cysteine replacements were grown in H1 minimal salts medium supplemented Unreacted AMS was removed by three washes in final buffer before the cells
with 30 mM succinate, 0.1% Casamino Acids, and ampicillin (100 ␮g ml⫺1) and were disrupted for 4 min at 100°C, immediately after the addition of SDS and
induced with 50 ␮M IPTG. Cysteine cross-linking using copper phenanthroline mPEG to a final concentration of 1% and 10 mM, respectively. The lysate was
was performed as described by Lee et al. (32) with the following modifications. incubated for another 15 min at 23°C before the reaction was stopped with 10 ␮l
Unless otherwise stated, the standard reaction was carried out at 23°C for various of SDS sample buffer containing 5% ␤-mercaptoethanol. Samples were boiled
time intervals (0, 2, 5, 10, and 15 min) and quenched with a stop solution for 5 min, run on SDS-PAGE, and Western blotted.
896 AMIN ET AL. J. BACTERIOL.

RESULTS
TM segments and secondary structure prediction of the
membrane anchor region. Twelve membrane protein prediction
programs were used to predict Aer membrane topology, but
there was no consensus among these algorithms. Nine pro-
grams forecasted just one transmembrane helix: MEMSAT,
residues 183 to 204 (25, 27); DAS, residues 168 to 203 (8);
PHDhtm, residues 173 to 197 (57, 58); TopPred, residues 167
to 187 (7, 64); TMHMM, residues 172 to 194 (30); TMpred,
residues 169 to 188 (21); HMMtop, version 2.0, residues 171 to
195 (63); TMAP, residues 164 to 192 (50, 51); and TMfinder,
residues 166 to 204 (12). Three of the programs predicted two
TM helices: PRED-TMR, residues 167 to 185 (TM1) and

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residues 187 to 204 (TM2) (49); SPLIT 4.0, residues 165 to 183
(TM1) and 187 to 207 (28); and SOSUI, residues 166 to 188
(TM1) and 196 to 218 (TM2) (20). The secondary structure for
this region was also analyzed. Jpred2 (9) and PSIpred (26),
which predict secondary structures most reliably from multiple
sequence alignments such as those from PSI-BLAST searches
(39, 40), predicted a helix-loop-helix for this membrane anchor
region.
In vivo cross-linking of single-cysteine replacements in the
membrane anchor. To analyze the structure of the membrane
anchor of Aer, we employed a cysteine disulfide cross-linking
approach (31, 43, 46). Wild-type Aer has three native cysteines
at positions 193, 203, and 253. A construct missing the three
native cysteines (C-less), encoding Aer-C193S/C203A/C253A
FIG. 2. Rates and extent of Aer cysteine cross-linking in intact cells
(38), was cloned into pTrc99A to create plasmid pMB1 (Ma-
after the addition of the oxidant copper phenanthroline. (A) The rate
terials and Methods). C-less Aer mediated aerotaxis on succi- of cross-linking for representative classes of cysteine replacements, as
nate swarm plates and was therefore functional. Plasmid pMB1 discussed in the text. Except for Aer-A184C (䊐) (left) and Aer-V187C
was used to introduce a single cysteine at desired positions, to (■) (left), the 10-min time point was in the linear range of the reaction
create a series of Aer mutants with cysteines that spanned for all cross-linking cysteine replacements, e.g., F182C (‚) and V209C
(Œ) (right). (B) Western blots showing (i) that the dimerization reac-
residues 163 to 210. This segment included the predicted mem- tion for Aer-A184C was nearly complete within 2 min (2⬘) and (ii) the
brane anchor and the bordering residues. All 48 single-cysteine presence of a dimer band in Aer-V187C before the addition of copper
replacements, including residues 163 to 210, mediated aero- phenanthroline (0⬘). (C) Representative Western blots comparing the
taxis in E. coli BT3312 (aer tsr) when inoculated on semisolid extent of cross-linking at 10 min, which was the time point chosen to
compare the extent of cross-linking for each cysteine replacement in
succinate agar, indicating that these cysteine replacements did the membrane anchor region. Abbreviations: M, monomer; D, dimer.
not significantly alter receptor function (data not shown).
The rates and extent of cysteine cross-linking in response to
the oxidant copper phenanthroline reflect the proximity of
these residues in cognate subunits of Aer and/or the flexibility
residue might be located in an oxidative environment. With
of the region (16, 22, 31). Once formed, the dimers can be
these exceptions (A184C and V187C), the 10-min time point
separated from non-cross-linked monomers by SDS-PAGE
was in the linear range for all constructs, and this incubation
under nonreducing conditions and visualized after Western
time was used for overall comparisons, as represented by the
blotting.
Initially, the rates of in vivo cross-linking after copper Western blots shown in Fig. 2C. The positive control, Aer-WT,
phenanthroline treatment at 23°C were determined. The rate contained three native cysteines, two of which can cross-link
plots for cross-linked cysteines fell into two major categories, (C203 in the membrane anchor and C253 in the HAMP do-
distinguished by the presence (residues 184 to 188, 205, 208, main) (38); the negative control, Aer-pMB1, had no cysteines.
and 209), or absence (residues 171, 173, 176, 182, 183, 191, 197, A contour plot generated by the PSA server at Boston Uni-
and 203) of visible cross-linking within 2 min. Once visible, versity (Fig. 3A) (60, 61, 66) is a convenient way to show the
however (with the exception of A184C and V187C) (Fig. 2A probability of the helix-loop-helix secondary structure in the
and B), the formation of the dimer product increased linearly membrane anchor region (37). The bar graphs below this con-
until approximately 15 min (see, e.g., F182C and V209C) (Fig. tour plot (Fig. 3B and C) are summaries of the cross-linking
2A). Residues A184C and V187C were unique in both the rate data from three or more independent experiments. At 23°C
and extent of cross-linking. A184C cross-linked most rapidly, (Fig. 3B), multiple residues cross-linked between cognate
and the reaction was nearly complete within 2 min (Fig. 2A and monomers (within a dimer) with no obvious periodicity. How-
B). V187C exhibited noticeable cross-linking in the absence of ever, there was strong cross-linking in consecutive residues
oxidant (Fig. 2A and B, 0-min time point), suggesting that this from F182C to V188C, consistent with close proximity and/or
VOL. 188, 2006 TOPOLOGY AND BOUNDARIES OF Aer IN E. COLI MEMBRANE 897

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FIG. 4. Surface accessibility of Aer cysteine replacements to the
sulfhydryl-reactive reagents 5-IAF and mPEG in membrane vesicles.
(A) SDS-PAGE gels showing the reactivity of strategic cysteine re-
placements towards 5-IAF. (Top) Negative fluorescent images of sam-
ples reacted with 5-IAF under native (N) and denaturing (D) condi-
tions for 5 min. (Bottom) The same bands stained with Coomassie blue
to estimate the total protein in each band. Note the unknown labeled
protein in the aer control lane under denaturing conditions. (B) Bar
graph summarizing the percent accessibility of cysteine replacements
to 5-IAF during incubations for 5 min at 23°C. The results are the
FIG. 3. Summary of cysteine cross-linking in Aer mutants at 23°C average of three or more independent experiments. The arrows at the
and 4°C. The extent of Aer dimers with cross-linked cysteines in the top of the graph represent the accessibility boundaries for mPEG at
membrane anchor demonstrates a central region of high proximity and 23°C (solid lines) and at 4°C (dotted lines). (C) Representative West-
flexibility, consistent with the PSA server secondary structure predic- ern blots showing the presence (residues 163, 184, 187, and 206) or
tion. (A) Contour plot of secondary-structure probabilities (PSA absence (residues 171, 189, 194, and 200) of a mobility shift in Aer
server) (60, 61, 66). Rows indicate the secondary structure state; col- (Aer-mp) after incubation of membrane vesicles with mPEG under
umns indicate each residue position. The probability of each structural native (N) conditions for 1 h. All cysteine replacements were PEGy-
state is depicted with contour lines in probability increments of 0.1. lated under denaturing (D) conditions.
The ␤-strand prediction value was ⬍0.1 and therefore is not included.
(B) Extent of in vivo cross-linking for all 48 cysteine replacements after
incubation of cells with copper phenanthroline for 10 min at 23°C.
(C) Extent of in vivo cross-linking for the same cells shown in panel B,
incubated with copper phenanthroline for 20 min at 4°C. Mapping membrane boundaries with 5-IAF and mPEG. To
map the boundaries of the Aer protein in the E. coli cytoplas-
mic membrane, we measured the surface accessibility of sub-
stituted cysteine residues by using sulfhydryl-reactive probes.
high flexibility in this region and supporting the loop structure The reagent 5-IAF is membrane impermeable and should not
predicted in the PSA contour plot (Fig. 3A). therefore react with integral membrane residues (3). Mixed
In reality, cross-linking between monomers might not rep- membrane vesicles expressing Aer single-cysteine replace-
resent close proximity between cognate residues but could ments at residues 163 to 210 were reacted with 5-IAF for 5 min
occur from random collisions of receptors diffusing laterally at 23°C, and the percentage of fluorescence associated with
through the lipid bilayer. To limit lateral diffusion, we repeated native Aer relative to denatured Aer was determined (Fig. 4).
the cross-linking analysis at 4°C (Fig. 3C), which is well below Representative gels showing the extent of 5-IAF labeling of
the lipid-phase transition temperature of approximately 18°C single-cysteine replacements under denaturing and native con-
in E. coli (45). To accommodate the decrease in kinetics at this ditions are shown in Fig. 4A. Coomassie blue-stained gels,
temperature, the reaction time was increased from 10 min to showing the relative levels of protein in each lane, are dis-
20 min. At 4°C, there was a low level of cross-linking in the played below the respective negative image of the same 5-IAF-
putative TM segments, but similar cross-linking (to that of labeled gel. There was a notable artifact under denaturing
23°C) in consecutive residues of the central loop (184 to 188), conditions with 5-IAF-treated membranes. The negative con-
consistent with flexibility in this region. Notably, A184C and trol lacking Aer (aer negative) showed apparent 5-IAF labeling
V187C still cross-linked at high levels, indicating close prox- under denaturing conditions and considerably less (but observ-
imity between neighboring residues at these positions. able) labeling under native conditions (Fig. 4A). This indicated
898 AMIN ET AL. J. BACTERIOL.

that another unidentified membrane protein(s) comigrated

with Aer. With this caveat in mind, we estimated the accessi-
bility of each cysteine to 5-IAF and have summarized these
data in Fig. 4B. As shown, there were large changes in acces-
sibility between residues 163 to 164, 183 to 184, 188 to 189, and
205 to 206, indicating that these regions are near the bound-
aries of the membrane and aqueous phases. From these data,
putative transmembrane segments would include residues 164
to 183 and 189 to 205.
To overcome the inherent background labeling with the FIG. 5. Pretreatment with AMS in intact cells blocks periplasmic
5-IAF probe, membrane boundaries were reanalyzed in mem- cysteine replacements from subsequent PEGylation. Cells were re-
brane vesicles by using another hydrophilic sulfhydryl-reactive acted with (⫹) and without (⫺) AMS for 45 min before being washed
reagent, mPEG (35). This reagent has an actual molecular and probed with mPEG under denaturing conditions as described in
the text. Aer-mPEG adducts (Aer-mp) showed a mobility shift. The

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mass of 5 kDa, but Aer-mPEG complexes exhibited an appar-
enlarged lanes in the lower panel are higher exposures taken from
ent molecular mass increase of ⬃10 kDa higher than that of different Western blots; these highlight replacements 184 and 187,
unreacted Aer (Fig. 4C), so the two forms could be separated which were reproducibly blocked by AMS.
by SDS-PAGE and analyzed on Western blots. As shown in the
representative Western blots of Fig. 4C, the Aer monomer and
Aer-mPEG forms were easily distinguishable (e.g., residues
184, 187, and 206), and there was no background signal from mic. The last membrane-embedded residue before the loop
membranes that lacked Aer. However, as previously reported was 181 (mPEG; 23°C) or 183 (5-IAF, 23°C; mPEG, 4°C) and
(38), an ever-present Aer proteolytic fragment was visible (Fig. the first membrane-embedded residue after the loop was 188
4C). (mPEG; 4°C), 189 (5-IAF; 23°C), or 190 (mPEG; 23°C). Taken
The lower background noise of this method allowed us to together, the maximum lengths of the transmembrane seg-
increase the protein loaded into each lane to maximize the ments include residues 164 to 183 for TM1 and 188 to 205 for
threshold at which we could visualize Aer-mPEG complexes. TM2.
As shown in Fig. 4C, residues predicted by the 5-IAF studies to The central flexible loop is periplasmic. Although the acces-
be membrane embedded, such as T189C, 193C, and A194C, sibility data and the predicted secondary structure of the mem-
showed no reactivity to mPEG in native membranes. However, brane anchor indicated that the central loop was periplasmic,
residues predicted to be near the membrane aqueous interface, we could not exclude unusual topologies wherein the flexible
such as A184C, V187C, and W206C, had high levels of reac- loop was cytoplasmic, since both sides of these vesicles are
tivity with mPEG. To eliminate the possibility of slow diffusion accessible to sulfhydryl reagents (3). To verify that the flexible
into the membrane, experiments were performed in parallel loop was periplasmic, we used an in vivo approach with AMS
with mPEG at 23°C and at 4°C for 1 h. At 4°C, mPEG does not (23, 35), a small hydrophilic sulfydryl-reactive reagent that can
cross membranes when incubated for 24 h (35). transverse the outer membrane porins but cannot cross the
A summary of the membrane boundaries determined by inner membrane. We pretreated intact cells with AMS to block
PEGylation at 23°C and 4°C are marked above the bar graph accessible periplasmic cysteines from subsequent reactions
in Fig. 4B. As expected, more residues were accessible to with mPEG under denaturing conditions.
mPEG at 23°C than at 4°C, and boundary demarcations were As shown in Fig. 5, residues A184C and V187C were com-
similar but not identical to those identified by 5-IAF. However, pletely blocked from PEGylation by AMS, indicating that
the combined data were consistent with a membrane anchor these residues are fully exposed to the periplasmic environ-
that has two membrane-spanning segments and a short ment. Thus, the central flexible loop was periplasmic and not
periplasmic loop. The N- and C-terminal boundaries would cytosolic. However, residues A185C and P186C, which were
represent the cytosolic membrane interface and the central accessible to both 5-IAF and mPEG in membrane vesicles,
loop boundaries would delineate the periplasmic membrane were not totally blocked by AMS (Fig. 5).
interface. The sequence of the Aer periplasmic loop is not critical for
Depending on the temperature and the particular sulfhydryl- function. The Aer-P186C replacement was functional, indicat-
reactive probe (Fig. 4B), the first membrane-embedded resi- ing that Pro186 was not absolutely required at this position. To
due after the N-terminal cytoplasmic domain was 164 (5-IAF; further investigate amino acid stringency at this position,
23°C), 165 (mPEG; 4°C), or 167 (mPEG; 23°C), and the last Pro186 was replaced by amino acid residues Arg, Ser, Phe, Trp,
membrane-embedded residue before the C-terminal cytoplas- Ala, and Asp. All replacements were functional in aerotaxis on
mic domain was 202 (mPEG; 23°C) or 205 (5-IAF, 23°C; semisoft succinate agar, indicating that this site was not critical
mPEG, 4°C). The higher accessibility of mPEG at 23°C sug- for activity (data not shown). The site was also permissive to
gested that the maleimide group of mPEG might penetrate short insertions, as a Ser-Gly-Ser replacement of Pro186 and
further into the membrane than 5-IAF. Whether this was due an Arg-Pro-Arg-Ile insertion between residues 185 and 186
to an inherent difference in hydrophobicity or caused by the (36) showed aerotaxis. The low level of amino acid stringency
difference in incubation time, which was 1 h for mPEG and 5 and permissiveness to insertions at this site are consistent with
min for 5-IAF, is not known. a region that is both near the membrane (for shielding hydro-
Similar temperature and reagent differences were evident phobic residues) and accessible to the periplasm (when accom-
for the central loop region, which was expected to be periplas- modating charged residues or insertions).
VOL. 188, 2006 TOPOLOGY AND BOUNDARIES OF Aer IN E. COLI MEMBRANE 899

DISCUSSION dimers unit. Preliminary evidence indicates that this is the case
(D. N. Amin, unpublished data). (iv) Membrane-spanning seg-
These data are consistent with a model in which the mem- ments might form a ␤-sheet rather than a helix. This scenario
brane anchor of Aer forms two membrane-spanning regions is unlikely, as secondary structure programs predicted ␣-heli-
(TM1 and TM2) flanking a central periplasmic loop. Such a ces for this region, and ␤-sheets are generally found in pore
model supports previous studies indicating that both the N- proteins, as well as being amphipathic. Helices form sponta-
terminal PAS domain and the C-terminal HAMP and signaling neously when a hydrophobic sequence inserts into a membrane
domains are cytosolic (11, 18, 62, 65, 68). bilayer, due to the free energy of hydrogen bonding between
Nine of the 12 membrane protein structure prediction pro- the polar backbone carbonyls and amide groups (15).
grams that were tested failed to predict two transmembrane Membrane boundaries were mapped in vitro by determining
segments for the 38 residues that were previously predicted to the accessibility of the cysteine replacements to the membrane-
form the Aer membrane anchor (1, 2, 52, 56). The best pre- impermeable sulfhydryl-reactive probes 5-IAF and mPEG.
diction methods can correctly identify all membrane helices in The mPEG probe had the advantage of specificity, as PEGy-

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only 50% to 70% of proteins with known structure (6). More- lated Aer could be discriminated from unmodified Aer on
over, developers may overestimate the accuracy of their meth- Western blots. For in vivo studies, we preblocked accessible
ods by 15% to 50% (5). PRED-TMR (49), SPLIT 4.0 (28), and cysteines with the periplasm-accessible, membrane-imperme-
SOSUI (20) were the only programs tested that predicted two able, sulfhydryl-reactive probe AMS before reacting solubi-
transmembrane segments, although SOSUI predicted spans lized cells with mPEG. The accessibility of residues in the
that were inconsistent with this study. Of note, a recent study flexible loop to 5-IAF and mPEG and the blocking of residues
in which the C termini of 601 E. coli inner membrane proteins 184 and 187 in vivo with AMS indicate that this loop is in the
were tagged with alkaline phosphatase and green fluorescent periplasm. The combination of strong cross-linking and acces-
protein correctly predicted a cytosolic location for both the sibility indicates that the loop is dynamic. Otherwise, an in-
N-terminal and C-terminal ends of Aer (11). verse relationship between cross-linking and accessibility
An in vivo cysteine cross-linking strategy was used to study would be expected. Interestingly, residues 185 and 186 were
the proximity of the transmembrane segments between cog- accessible to 5-IAF and mPEG but not to AMS. One possible
nate monomers within the Aer dimer (31, 38, 43, 46). Several explanation for this difference is that AMS is more selectively
consecutive residues (182 to 188) flanking Pro186 in the cen- excluded from the membrane surface than is 5-IAF or mPEG.
tral region of the membrane anchor cross-linked strongly at In this case, residues 185 and 186 would be proximal to the
23°C (Fig. 3B), indicating that this segment was highly flexible. periplasm but would not protrude into the periplasm. Alter-
Moreover, residues 184 to 187 cross-linked strongly at 4°C natively, the presence of a membrane potential in whole cells
(Fig. 3C), suggesting that the cross-linking was due to proxim- or other changes occurring during the preparation of mem-
ity between monomers and not to random collisions from lat- brane vesicles could alter the local topology and shield these
erally diffusing receptors in the membrane. In E. coli, the residues from the aqueous phase.
lateral diffusion of membrane proteins decreases markedly be- Accessibility studies indicated that the membrane cytosolic
low 18°C, which is the lipid-phase transition temperature (45). boundaries lay between residues 164 and 167 for TM1 and
An example of this temperature effect was shown for lactose between residues 202 and 205 for TM2. Residues 163 and 206
permease, where strong cross-linking that occurred from ran- were accessible to all probes under all conditions (23°C and
dom collisions between monomers at room temperature was 4°C). Notably, residues 163 and 206 are tryptophans in native
markedly inhibited at 0°C during 1-h incubations (17). Aer. Tryptophans are well known for stabilizing membrane-
Unlike the central loop region, there was sparse cross-link- aqueous boundaries (13, 67). Given their hydrophobic nature,
ing in the TM1 and TM2 segments. Moreover, residues that it is likely that these native tryptophan residues reside at the
cross-linked did not show the periodicity one would expect for membrane boundary interface rather than protrude into the
close interactions between cognate helices (33). Several expla- cytosol, as occurred for their cysteine replacements.
nations for low cross-linking levels and lack of periodicity are From the cysteine accessibility data, the maximum lengths of
possible. (i) The relative tilt of the cognate transmembrane the transmembrane regions would include 20 residues for TM1
helical segments, driven by the solvation of side chains at the (from 164 to 183) and 18 residues for TM2 (from 188 to 205).
membrane boundaries (54, 55), may limit cross-linking. (ii) The value for TM2 is close to the average length (17.7 residues
The nearest helix-helix interactions might occur through TM1- or 27 Å) (19) for a membrane helix that spans the hydrophobic
TM2 or TM1-TM2⬘ segments rather than via TM1-TM1⬘ and part of the bilayer. The absolute minimum lengths for these
TM2-TM2⬘ segments. That said, the sequence of amino acids segments would include 15 residues for TM1 (167 to 181) and
in the transmembrane segments of Aer did not show periodic 13 residues for TM2 (190 to 202). These lengths are untenable
variations in polarity, as one might expect for TM-TM inter- if a helical structure is assumed, as they would not span the
actions. This is consistent with the low level of sequence con- membrane (27 Å). As stated for the AMS probe, variations in
servation found in the membrane anchor region. In general, accessibility between 5-IAF and mPEG sulfhydryl probes may
residues facing outward toward the lipids are considerably also represent differences in the ability of these probes to
more hydrophobic than those facing inward, toward the other penetrate the membrane surface. The estimates for the maxi-
helix(es) in the membrane (53), opposite of that seen in aque- mum TM1 and TM2 lengths appear reasonable and, as stated,
ous environments. (iii) Helix-helix interactions may not be are supported by the presence of tryptophans at residues 163
exclusively intradimeric; some residues may cross-link within a and 206.
dimer while others cross-link between dimers in a trimer of The central loop between TM1 and TM2 contains a non-
900 AMIN ET AL. J. BACTERIOL.

conserved proline residue; prolines are established helix break- 12. Deber, C. M., C. Wang, L. P. Liu, A. S. Prior, S. Agrawal, B. L. Muskat, and
A. J. Cuticchia. 2001. TM Finder: a prediction program for transmembrane
ers in globular proteins (34). In model membrane systems, a protein segments using a combination of hydrophobicity and nonpolar phase
single proline residue can change a 40-mer hydrophobic pep- helicity scales. Protein Sci. 10:212–219.
tide from a single membrane-spanning helix into two mem- 13. Draheim, R. R., A. F. Bormans, R. Z. Lai, and M. D. Manson. 2005. Tryp-
tophan residues flanking the second transmembrane helix (TM2) set the
brane-spanning helices (44). However, in membrane segments, signaling state of the Tar chemoreceptor. Biochemistry 44:1268–1277.
prolines may often stabilize the helical structure (34), and 14. Ehrmann, M., D. Boyd, and J. Beckwith. 1990. Genetic analysis of mem-
bends within membrane regions generally require more than brane protein topology by a sandwich gene fusion approach. Proc. Natl.
Acad. Sci. USA 87:7574–7578.
one proline unless a glycine residue is spaced four residues 15. Eilers, M., S. C. Shekar, T. Shieh, S. O. Smith, and P. J. Fleming. 2000.
away (24). Previously, we found that Aer was functional after Internal packing of helical membrane proteins. Proc. Natl. Acad. Sci. USA
a tetrapeptide (RPRI) had been inserted between residues 97:5796–5801.
16. Falke, J. J., A. F. Dernburg, D. A. Sternberg, N. Zalkin, D. L. Milligan, and
Ala185 and Pro186 (36). In the present study, we found that D. E. Koshland, Jr. 1988. Structure of a bacterial sensory receptor. A site-
amino acid replacements at Pro186 in Aer did not abolish the directed sulfhydryl study. J. Biol. Chem. 263:14850–14858.
function. In addition to the Cys replacement, Aer was func- 17. Guan, L., F. D. Murphy, and H. R. Kaback. 2002. Surface-exposed positions
in the transmembrane helices of the lactose permease of Escherichia coli

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tional when Pro186 was replaced by polar (S), charged (D and determined by intermolecular thiol cross-linking. Proc. Natl. Acad. Sci. USA
R), or aromatic (F and W) residues, as well as with a tripeptide 99:3475–3480.
18. Herrmann, S., Q. Ma, M. S. Johnson, A. V. Repik, and B. L. Taylor. 2004.
(SGS). These data and the fact that Pro186 is not conserved in PAS domain of the Aer redox sensor requires C-terminal residues for native-
other Aer receptors indicate that proline is not necessary to fold formation and flavin adenine dinucleotide binding. J. Bacteriol. 186:
form the two transmembrane segments. Moreover, since the 6782–6791.
19. Hildebrand, P. W., R. Preissner, and C. Frommel. 2004. Structural features
loop was permissive to short peptide inserts, the region must of transmembrane helices. FEBS Lett. 559:145–151.
not be directly involved with signaling. 20. Hirokawa, T., S. Boon-Chieng, and S. Mitaku. 1998. SOSUI: classification
In summary, the Aer membrane anchor in E. coli forms two and secondary structure prediction system for membrane proteins. Bioinfor-
matics 14:378–379.
transmembrane segments that flank a central short periplasmic 21. Hofmann, K., and W. Stoffel. 1993. TMbase—a database of membrane
loop. The loop itself does not appear to be involved in signal spanning protein segments. Biol. Chem. Hoppe-Seyler 374:166.
transduction. Whether the function of the membrane anchor is 22. Hughson, A. G., and G. L. Hazelbauer. 1996. Detecting the conformational
change of transmembrane signaling in a bacterial chemoreceptor by mea-
to localize Aer to the membrane, maintain registry between suring effects on disulfide cross-linking in vivo. Proc. Natl. Acad. Sci. USA
N-terminal PAS and C-terminal signaling regions, or be ac- 93:11546–11551.
tively involved in signaling like the MCPs (13, 42) remains to 23. Iwaki, S., N. Tamura, T. Kimura-Someya, S. Nada, and A. Yamaguchi. 2000.
Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the
be determined. Tn10-encoded metal-tetracycline/H⫹ antiporter reveals a permeability bar-
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ACKNOWLEDGMENTS
24. Javadpour, M. M., M. Eilers, M. Groesbeek, and S. O. Smith. 1999. Helix
We thank Maxwell Brandon for creating pMB1, Gordon Harding packing in polytopic membrane proteins: role of glycine in transmembrane
for developing the PEGylation protocol, and Kylie Watts for critical helix association. Biophys. J. 77:1609–1618.
25. Jones, D. T. 1998. Do transmembrane protein superfolds exist? FEBS Lett.
analysis and helpful discussions. We are grateful to Sheena Fry and
423:281–285.
Nathan Abraham for technical assistance. 26. Jones, D. T. 1999. Protein secondary structure prediction based on position-
This work was supported by grants from Loma Linda University to specific scoring matrices. J. Mol. Biol. 292:195–202.
M.S.J. and the National Institute of General Medical Sciences 27. Jones, D. T., W. R. Taylor, and J. M. Thornton. 1994. A model recognition
(GM29481) to B.L.T. approach to the prediction of all-helical membrane protein structure and
topology. Biochemistry 33:3038–3049.
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