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J Biol Chem, Vol. 273, Issue 47, 31113-31118, November 20, 1998
From the The btuB gene product from
Escherichia coli is a 66.5-kDa integral outer membrane
protein required for high-affinity uptake of cyanocobalamin and the
translocation of E group colicins and colicin A. Efficient purification
of overexpressed BtuB containing stoichiometric levels of bound
lipopolysaccharide has been achieved through the extraction of the
outer membrane with nonionic detergent followed by ion-exchange
chromatography. Analysis of far UV circular dichroism spectra indicates
a predominantly The outer membrane of Escherichia coli and other
Gram-negative bacteria has evolved to exclude harmful agents present in
the surrounding environment while maintaining the ability to
internalize required nutrients of low abundance. Although general
barrier function is largely provided by the lipopolysaccharide
comprising the outer leaflet of this asymmetric bilayer (1, 2), the complexities of nutrient uptake are addressed by a variety of integral
membrane proteins that operate by passive diffusion, facilitated
diffusion, or active transport (3, 4). Bacterial porins form trimeric,
aqueous-filled channels that direct passive or facilitated diffusion of
hydrophilic compounds up to a molecular weight limit of approximately
600 (3). Extensive biochemical and electrophysiological
characterization (5), together with high resolution structure
determination of both porin types (6, 7), has provided insight into the
manner in which a membrane-spanning The acquisition of vitamin B12 and iron-bearing
siderophores presents additional complications for E. coli
cells due to their relative low abundance and molecular masses that
exceed the diffusion limit of porin channels (8). The uptake of such
compounds involves a related family of outer membrane,
TonB-dependent receptors (e.g. FhuA, FepA, Cir)
that selectively bind ligands with high affinity and derive the
metabolic energy required for transport through a poorly understood
coupling with the energy-transducing TonB complex (9). Structural
models derived from sequence analysis (10) and biochemical studies (11)
have long suggested that TonB-dependent receptors consist
of The btuB gene product from E. coli is a
66,412-dalton, TonB-dependent receptor essential for
high-affinity uptake of cyanocobalamin that also serves as a receptor
for the E group colicins, colicin A, and phage BF23 (16). Although the
ligand binding properties of purified receptor have been described
previously (17, 18), and insights into both ligand binding (19, 20) and
the mechanism of cobalamin transport (21, 22) have resulted from the
characterization of BtuB mutants incorporated into the outer membrane,
the lack of a purification procedure with a high yield has precluded a more detailed understanding of receptor properties. The present work
describes procedures for overexpression and purification of the
E. coli vitamin B12 receptor in quantities
sufficient for characterization and crystallization trials and provides
information on the secondary structure, aggregation state, and ligand
binding properties of the purified, protein-detergent complex.
Reagents--
Mega-10 (Anagrade) and octylglucoside (Sol grade)
were obtained from Anatrace, Inc. Tryptone and yeast extract were
purchased from Difco, DEAE-Sepharose CL-6B and Sephadex G-15 were
purchased from Sigma and Pharmacia, respectively, and low molecular
weight protein standards were purchased from Bio-Rad. Vitamin
B12 and LPS1
(E. coli serotype 055:B5 and EH100) were obtained from
Sigma. Colicin E1 (23) and colicin E3 (24) were purified as described previously.
Overexpression of BtuB--
The outer membrane protein-deficient
E. coli strain TNE012 (K12 tsx Outer Membrane Isolation and Extraction--
Frozen cell pellets
from 4 liters of culture were resuspended to approximately 20 ml with
200 mM Tris, pH 7.5 (the pH of all buffers was adjusted at
room temperature). Lysozyme (30 mg dissolved in 200 mM
Tris) was added to stirred cells along with 23 ml of a 1 M
sucrose, 5 mM EDTA solution prepared by dissolving sucrose in 200 mM Tris and adding EDTA from a 100 mM
stock, pH 7.5. After a 1-min incubation, 45 ml of distilled water and
800 µl of phenylmethylsulfonyl fluoride (17.5 mg/ml) and
N-tosyl-L-phenylalanine chloromethyl ketone (3.5 mg/ml) in ethanol were added. Cells were stirred for 30 min before the
addition of 1.38 ml of 1 M MgSO4 and
deoxyribonuclease (10 mg dissolved in 200 mM Tris). After a
15-min incubation, 9.6 ml of 20% Triton X-100 were added, and the
cells were incubated for an additional 15 min. The aforementioned steps
were conducted at 4 °C. The resulting cell extract was centrifuged
for 45 min at 144,000 × g in a Ti60 rotor at 4 °C.
The resulting pellets were rehomogenized in 50 ml of ice-cold 50 mM Tris and 5 mM EDTA (Buffer A) to which 500 µl of the phenylmethylsulfonyl
fluoride/N-tosyl-L-phenylalanine chloromethyl
ketone solution had been added, and centrifugation was carried out as
described above. All subsequent steps were conducted at room
temperature. Centrifuge pellet fractions were further washed in 25 ml
of Buffer A and 2% Triton X-100 and centrifuged at 20 °C under the
same conditions described above. The resulting pellet fractions were
rehomogenized in 20 ml of Buffer A, brought to 1% Mega-10 by the
addition of a 10% stock solution, and nutated for 20 min before
centrifugation for 30 min at 177,500 × g at 20 °C.
The resulting supernatant fraction was saved, and BtuB was isolated as
described below. The inclusion of 0.25% LIS during extraction
effectively eliminated proteolytic degradation, if it was observed
during the extraction of BtuB.
BtuB Purification--
The supernatant fraction from the Mega-10
extract was diluted into 50 ml of Buffer A, 1% Mega-10, and 0.07% LIS
and loaded onto a 10-ml DEAE-Sepharose CL-6B column. The column was
washed with 20 ml of Buffer A, 0.35% Mega-10, and 0.05% LIS and
eluted with 100 ml of 0-700 mM LiCl gradient in Buffer A,
0.35% Mega-10, and 0.05% LIS. Due to the intense UV absorption of
LIS, the broad BtuB peak eluted throughout the latter half of the
gradient was detected by Coomassie Blue staining (~15 min) of the
fractions spotted onto Whatman #1 filter paper, followed by destaining
with 20% methanol and 10% acetic acid. Pooled fractions were
concentrated to approximately 2 ml with a Centriprep-50, diluted into
40 ml of Buffer A and 1% Mega-10, and loaded onto a 2-ml
DEAE-Sepharose CL-6B column. Detergent exchange was achieved by washing
the column with 10 ml of Buffer A and 1% OG, followed by elution with
70 ml of 0-450 mM LiCl gradient in Buffer A and 1% OG.
Pooled receptor fractions were concentrated to approximately 2 ml in a
Centriprep-50, and buffer exchange was carried out with a 10-ml
Sephadex G-15 column equilibrated with 15 mM
NaH2PO4 or 10 mM Tris and 1% OG, pH 7.5. Fractions were desalted and concentrated with a Centricon-100 and stored at SDS-PAGE and Gel Staining Methods--
BtuB fractions were
routinely assessed for purity using a Tris-Tricine gel system (26) with
stacking and resolving gels of 3 and 12.5%, respectively. Receptor
conformation could be evaluated qualitatively by SDS-PAGE on a Phast
Gel system (Pharmacia) using 12.5% acrylamide gels. Protein was
detected by staining with either silver (27) or Coomassie Brilliant
Blue (28). Bacterial lipopolysaccharide was resolved on the
Tris-Tricine system described above and detected by
carbohydrate-sensitive silver staining (29); development was terminated
by a brief incubation in 20%
Na2S2O3. The LPS profile from the
E. coli K12 derivative used for overexpression was evaluated by SDS-PAGE after microextraction and proteinase K digestion (30). The
LPS content of BtuB preparations was estimated by densitometry of
silver-stained gels with standard curves generated from E. coli EH100 LPS, which contains lipid A and core sugars but lacks O-antigen, similar to the E. coli K12 derivative used for
BtuB overexpression (Ref. 2; Fig. 2). Staining of LPS was conducted such that all species remained in the linear densitometric range.
Circular Dichroism--
All measurements were acquired at room
temperature using a JASCO-600 spectropolarimeter. Quartz cells with a
path length of 0.2 mm were used for far UV measurements. Spectral
parameters were as follows: (a) time constant, 2 s;
(b) scan speed, 20 nm/min; (c) scanning
increment, 0.1 nm; and (d) spectral bandwidth, 2 nm. Spectra
were recorded in 15 mM NaH2PO4 and
1% OG titrated to pH 7.5 with NaOH at protein concentrations of
approximately 0.5 mg/ml, and the buffer background was subtracted. Far
UV CD spectra of BtuB were analyzed for secondary structure information by the methods of Provencher and Glockner (31) and Chang et al. (32). The mean values of secondary structure content obtained from the two methods are reported.
Analytical Ultracentrifugation--
Sedimentation equilibrium
and sedimentation velocity measurements were performed in the Beckman
OPTIMA XL-A analytical ultracentrifuge (AN60Ti rotor). BtuB dissolved
in 15 mM NaH2PO4 and 1% OG, pH 7.5, was analyzed at concentrations of 0.5 mg/ml for velocity experiments and 0.35, 0.75, and 1.5 mg/ml for equilibrium experiments. Equilibrium and velocity runs were conducted in a 12-mm double sector
cell at 20 °C, and absorbance was monitored at 275 nm. Velocity
studies were performed at 50,000 rpm with sedimentation boundaries
scanned at 10-min intervals. Sedimentation equilibrium studies were
performed for 30 h at 10,000 rpm. The best fit to the
sedimentation equilibrium data was analyzed with the program SEDEQ1B
(A. P. Minton; minton{at}helix.nih.gov), and the velocity data were
analyzed by both the Svedberg (33) and the DCDT method (34). The
partial specific volume of BtuB (0.726 cm3/g) was
calculated from the amino acid composition. A partial specific volume
of 0.859 cm3/g was used for octylglucoside (35). Molecular
masses were generated for both the protein-detergent complex and the
protein component alone, estimating the detergent bound to the protein
at 0.5, 1.0, and 1.5 g/g (35). Sedimentation velocity experiments were
conducted as described above after the preincubation of BtuB (0.5 mg/ml) with equimolar levels of colicin E3 to directly demonstrate the formation of the BtuB-colicin E3 complex. The binding of vitamin B12 was measured in a similar fashion by simultaneously
monitoring the sedimentation of protein (275 nm) and cyanocobalamin
(358 nm) after the preincubation of the vitamin (15 or 30 µM) with BtuB (15 µM) solubilized in 10 mM Tris, 5 mM CaCl2, and 1% OG, pH
7.5.
Colicin Neutralization--
Receptor activity was also evaluated
as the ability of purified BtuB to protect susceptible cells from the
killing action of colicin E1 or E3. The sensitive E. coli
strain K17DE3 (pT7-7) was grown to mid-log phase, and 150-µl
aliquots were spread on 2YT Petri dish plates containing 100 µg/ml
ampicillin. The plates were dried for 30 min at room temperature before
the sample addition. The samples assayed for cytotoxicity were diluted
to a final volume of 20 µl and preincubated for 20 min at room
temperature, and 7.5 µl were subsequently applied to the plates
described above. The plates were incubated overnight at 37 °C after
the applied samples had dried, and colicin activity was evaluated as
the ability to maintain a clear zone within a lawn of growing cells.
All dilutions and reagents were prepared in 20 mM Tris and
1% OG, pH 7.5. Vitamin B12 was added to BtuB 10 min before
the addition of colicin in studies that assessed its effects on
receptor function. Control experiments indicated that colicin was the
agent responsible for cell killing under all conditions examined.
Overexpression and Purification--
The use of E. coli
TNE012 as an expression strain for BtuB purification has been described
previously (17). However, an alternate procedure was necessary for the
isolation of protein in quantities sufficient for characterization and
crystallization trials. Overexpression from plasmid pJC3 coupled with
the extraction of outer membranes with the nonionic detergent Mega-10
dramatically increased the amount of starting material for purification
(25). Although prolonged cell growth may further enhance the expression levels, proteolytic degradation had been noted under such conditions, resulting in the detection of 20- and 40-kDa BtuB degradation products
by SDS-PAGE. Co-purification of the degradation products likely
resulted from a stable association between the fragments throughout the
purification (data not shown).
The purity of BtuB after various stages of extraction and purification
is shown in Fig. 1. Initial DEAE
chromatography was facilitated by the presence of 0.05% LIS during
column loading and elution, due to its ability to both inhibit
observable proteolytic degradation and increase receptor purity by
shifting the elution of BtuB to a later portion of the gradient. A
second DEAE step was carried out for LIS removal, detergent exchange,
and final purification. Pooled fractions from this second DEAE column
contain a receptor species that is pure with respect to the protein
(Fig. 1, lane 4) yet contains bound lipopolysaccharide (Fig.
2). Densitometry after SDS-PAGE and
carbohydrate-sensitive silver staining using E. coli EH100
LPS for the generation of standard curves indicated that preparations
contain approximately 2 ± 1 mol bound LPS/mol BtuB (data not
shown).
Secondary Structure Analysis by Circular Dichroism and
SDS-PAGE--
Far UV CD spectra (190-250 nm) of BtuB
solubilized in the nonionic detergent
The characterization of the TonB-dependent receptors FhuA,
FepA, Cir (11, 36), and a variety of porins by SDS-PAGE (5) revealed
heat-modifiable migration patterns that resulted in an underestimation
of Mr for protein exposed to electrophoresis
sample buffer in the absence of heating. Such behavior has been
proposed to stem from a compact, SDS-resistant, Analytical Ultracentrifugation--
Octylglucoside-solubilized
BtuB was characterized by sedimentation equilibrium and sedimentation
velocity to ascertain the oligomeric state and ensure the
monodispersity of the protein-detergent complex. The analysis of
equilibrium data yields plots of ln A versus r2
that are linear from the meniscus to the cell bottom (Fig.
4A), indicating a homogenous
preparation characterized by an aggregation state that is independent
of sample concentration (0.35-1.5 mg/ml). Sample homogeneity is
further demonstrated by the normal distribution of the sedimentation
values obtained from the velocity studies (Ref. 34; Fig.
4B). The minor inhomogeneity observed at low S values is
likely a result of light scattering from the slowly sedimenting
octylglucoside micelles (Fig. 4B). From the slope of the ln
A versus r2 plot (Fig. 4A) and the
sedimentation velocity data (Fig. 4B) analyzed according to
Ref. 33, a sedimentation coefficient of 4.7 S and a molecular weight of
89,000 were calculated for the protein-detergent complex. The molecular
weight of 89,000 includes an appreciable contribution from bound
detergent. Calculations based on the assumption of a protein-detergent
complex with detergent bound at levels of 1 g/g protein (13, 35) imply
a contribution of approximately Mr 60,000 for
the protein component alone, indicating that BtuB was purified as a
monomer. Similar calculations with estimates of detergent bound at a
ratio of 0.5 g/g (approximately the molecular weight of a detergent
micelle) or 1.5 g/g protein result in only a 20% difference in the
calculated molecular weight, which is consistent with the conclusion
that purified BtuB is monomeric.
Ligand Binding Characteristics of Purified BtuB--
The initial
binding to the receptor in the outer membrane is an obligate step in
colicin action. The formation of a high-affinity complex between
colicin and purified receptor will decrease the concentration of free
colicin, resulting in the protection of otherwise susceptible strains.
The ligand binding characteristics of isolated BtuB were qualitatively
evaluated by testing its ability to eliminate the cytotoxic effects of
colicins E1 and E3. The preincubation of colicin E3 (16 nM)
with increasing concentrations of BtuB eliminated observable cell
killing once the receptor was provided in modest stoichiometric excess
(22.5 nM; data not shown). In the absence of BtuB,
cytotoxicity was apparent at a colicin E3 concentration of 0.16 nM, indicating that detergent-solubilized receptor retains
high affinity for this particular ligand (Kd < 1 nM). In contrast, the preincubation of colicin E1 (17.5 nM) with a 15-fold molar excess of BtuB conferred no
observable protection. Vitamin B12 did not prevent the
neutralization of colicin E3 by BtuB, even when it was present at
approximately a 200-fold molar excess over the colicin.
The ability of detergent-solubilized BtuB to bind both vitamin
B12 and colicin E3 was directly demonstrated by
sedimentation velocity analysis. Preincubation with stoichiometric
levels of BtuB (15 µM) shifted the sedimentation profile
of vitamin B12 (Fig.
5B) to that observed for the
isolated protein-detergent complex (Fig. 5A). Similar
experiments carried out with a vitamin B12:BtuB molar ratio
of 2:1 revealed that approximately half of the vitamin does not
sediment to any significant extent, as indicated by a nonsedimenting
baseline equal to approximately half the total amplitudes (Fig.
5C). This implies a binding stoichiometry of approximately
1:1. In the case of colicin E3, the addition of equimolar
concentrations of colicin (Fig. 6, traces
e-h, measured at the same 10-min intervals as
a-d) converts BtuB to a more rapidly migrating species with
a sedimentation coefficient of 6.3 S. It is of interest to compare the
sedimentation behavior of BtuB (4.7 S; Mr
~89,000) and the BtuB-colicin E3 complex (6.3 S;
Mr ~150,000 upon the addition of colicin E3
(Mr 61,000) to the Mr
89,000 receptor-detergent complex) with other characterized
protein-detergent complexes of similar size: cytochrome c
oxidase in C8E45 (4.1 S;
Mr ~84,000; Ref. 38),
(Na+K+)-ATPase in C12E8
(6.5 S; Mr 141,000; Ref. 39), and monomeric cytochrome b6f complex in
dodecylmaltoside (6.3 S; Mr 149,000; Ref.
40).
Compared with previous studies (17, 18), the present study on the
E. coli vitamin B12 receptor has contributed the
following: (a) the receptor has been purified to apparent
homogeneity with greater than 50-fold higher yields; (b) the
stoichiometry and homogeneity of LPS associated with BtuB have been
determined after purification; (c) the secondary structure,
which was previously based on prediction alone, has been determined by
a comprehensive far UV circular dichroism analysis; and (d)
the oligomeric state of isolated detergent-solubilized BtuB was
rigorously determined.
Purified BtuB: A Potential Model System for Membrane Protein
Structure Determination and Receptor-Ligand Interactions--
The
availability of a rapid and convenient procedure for the purification
of BtuB makes it an attractive system to study the characteristics of
the TonB-dependent receptor family as well as the general
principles of membrane protein structure, receptor-ligand interactions,
and energy-dependent transport phenomena. The isolation scheme presented in this work not only results in higher yields but
also avoids the laborious and unreliable process of affinity chromatography on colicin E3-Sepharose (17). The purified receptor contains LPS, as previously observed during the purification of other
outer membrane proteins (36, 41). It will be of interest to evaluate
the effects of delipidation on receptor properties because this species
appears tightly bound relative to the situation with bacterial porin in
which LPS could be removed through ion-exchange chromatography and
ultrafiltration (42). Repeated desalting with a Centricon-100 did not
eliminate contaminating LPS from BtuB preparations. Similar experiments
with E. coli EH100 LPS indicated that LPS should be removed
under such conditions if it is not bound to the receptor (data not
shown). The removal of associated LPS was previously shown to be
necessary for the crystallization of porin (42). However, the low
LPS:BtuB stoichiometry (2 ± 1:1), combined with an LPS species
that appears homogenous in size (Fig. 2), raises the possibility that
the bound LPS does not provide a major source of microheterogeneity.
Spectroscopic and SDS-PAGE Analysis of the Secondary Structure and
Conformational Changes--
Sequence analysis of both porins and
TonB-dependent receptors reveals an absence of hydrophobic
stretches of sufficient length to span the bilayer as independent
Resistance to denaturation by SDS results in a compact,
faster-migrating species during SDS-PAGE if the samples are not boiled before electrophoresis, providing a simple, rapid means of evaluating receptor conformation. This property may prove of particular value for
crystallization trials, allowing insights into the effects of prolonged
storage and environmental conditions on protein conformation. The CD
spectrum of BtuB obtained after heat denaturation in the presence of
OG/SDS mixed micelles is peculiar. The expectation of a single peak of
negative ellipticity centered between 195 and 200 nm for a fully
denatured protein (37), coupled with the relatively weak ellipticity of
the two negative transitions and the well-characterized ability of SDS
to induce Oligomeric State of Purified BtuB--
In contrast to the
situation with a variety of bacterial porins that associate to form
highly stable trimers, the literature concerning the oligomeric state
of the TonB-dependent receptors FhuA, FepA, and BtuB is
less clear. The characterization of detergent-solubilized FhuA by
sedimentation equilibrium and sedimentation velocity indicated that it
was purified as a monomer (13, 36), although further analysis of the
ligand binding properties and the ability to cross-link a certain
fraction protein into dimers and trimers led to the proposal of
transient oligomerization in solution (36). For the
TonB-dependent receptor FepA, Western blot analysis of
outer membrane fractions after solubilization with lithium dodecyl
sulfate and LDS-PAGE raised the possibility that it was extracted as a mixed population of monomers and trimers (15), although such experiments cannot rule out the possibility of stable hetero-oligomer formation. For BtuB, the possibility of quaternary structure or hetero-oligomer formation within the context of native outer membranes remains to be explored. However, the centrifugation analysis carried out in the present study clearly indicate that the receptor was purified as a monomer. A combination of BtuB and OmpF serving as the
optimal colicin A receptor (45) suggests at least a transient association between these two species during the colicin
translocation process.
Ligand Binding Characteristics of Purified BtuB--
The isolation
scheme described in this work results in a receptor species that
retains the ability to bind both vitamin B12 and colicin
E3. Vitamin B12 had no observable effect on the ability of
the receptor to neutralize colicin E3 in colicin cytotoxicity assays
(see "Results"), even though common binding sites may be inferred
from studies demonstrating the ability of the vitamin to protect cells
against the action of colicins E1 and E3 in vivo (46).
Considering the lower receptor/ligand concentrations used for the
colicin cytotoxicity assays (nanomolar concentration, colicin;
nanomolar concentration, receptor; micromolar concentration, vitamin)
compared with those used for the sedimentation velocity studies
(micromolar concentrations were used for all components), these results
suggest that the affinity of BtuB for vitamin B12 is
somewhat decreased after purification. Preliminary sedimentation velocity studies indicate that colicin E1 and vitamin B12
bind to BtuB in a competitive fashion (data not shown), further
suggesting that the aforementioned ligands either share a common
binding site or that the binding of one ligand results in a loss of
determinants required for the recognition of a second ligand at a
discrete site.
The inability of purified receptor to neutralize colicin E1 is
consistent with prior observations suggesting that BtuB may be only one
component of the colicin E1 receptor with the additional, required
determinants removed during purification (17). The tolC gene
product codes for a porin-like outer membrane protein uniquely required
in the mechanism of colicin E1 (47). The possibility exists that an
association between BtuB and TolC is required for the high-affinity
binding of colicin E1, analogous to the requirement of both BtuB and
OmpF as the optimal colicin A receptor (45).
We thank F. M. Richards for helpful
discussions in the early stages of the project, M. Lindeberg and C. V.
Stauffacher for subsequent helpful discussions, and J. Hollister for
assistance with the manuscript.
*
These studies were supported in part by National Institutes
of Health Grant GM-18457 (to W. A. C.) and National Institutes of
Health Biophysics Training Grant GM-08296 (to R. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by National Institutes of Health Grants 4652115 (C. V.
Stauffacher) and GM 45756 (V. J. Davisson).
The abbreviations used are:
LPS, lipopolysaccharide; CD, circular dichroism; LIS, lithium
3,5-diiodosalicylate; OG,
Purification and Characterization of Monomeric Escherichia
coli Vitamin B12 Receptor with High Affinity for
Colicin E3*
,§,
Department of Biological Sciences, Purdue
University, West Lafayette, Indiana 47907 and the ¶ Department
of Biochemistry, Brandeis University, Waltham, Massachusetts 02254
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-sheet secondary structure (76 ± 4%) with a
low 
-helical content (15 ± 3%), providing the first direct
evidence for secondary structure models derived from sequence and
hydropathy analysis. Characterization of the octylglucoside-solubilized
receptor by sedimentation equilibrium and sedimentation velocity
analysis reveals a monodisperse protein-detergent complex of
approximately 89 kDa with a sedimentation coefficient of 4.7 S which,
after correction for bound detergent, indicates that BtuB is purified
as a monomer. BtuB binds vitamin B12 with a stoichiometry
of approximately 1:1, as observed by a shift in the sedimentation
profile of the vitamin to the much faster velocity observed for the
protein-detergent complex. The preincubation of colicin E3 with
stoichiometric levels of BtuB protects susceptible strains from the
lethal effects of the colicin and results in a complex with a
sedimentation coefficient appropriate for a BtuB-detergent-colicin E3
complex, demonstrating that monomeric BtuB retains high affinity for
this particular ligand after isolation.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-barrel architecture serves to
overcome the energetic problems associated with the transport of polar
solutes through the membrane bilayer. The comparison of passive
diffusion channels with those functioning by facilitated diffusion
illustrates the nature of modifications to the basic porin fold that
confer specificity to the transport process (6).
-barrel transmembrane domains coupled with extramembrane loop
regions that are extensive compared with those observed in porin
structures. The recent spectroscopic analysis of FhuA (12, 13) provided
the first direct evidence for a predominantly -sheet secondary
structure for detergent-solubilized receptor retaining the ability to
bind both bacteriophage T5 and ferrichrome. Insight into the function
of FhuA (14) and FepA (15) has been obtained through
electrophysiological characterization of deletion mutants that, in
contrast to the closed state observed for the wild-type protein, form
relatively large, permanently open, aqueous diffusion pores after the
excision of surface-exposed loops thought to be critical for channel
gating. Although the monomeric state observed for detergent-solubilized
FhuA (13) contrasts with porin quaternary structure (6), the available data has prompted the concept of TonB-dependent receptors
as gated porins comprised of large, aqueous pores buried within the
outer membrane. These pores are apparently coupled to extensive loop regions that provide the additional determinants required for function
as selective, energy-dependent solute transporters.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ompA ompB) was used for overexpression
from plasmid pJC3 (25). Cells grown at 37 °C to an
OD600~1.0 in RB medium (10 g of bactotryptone and 5 g of yeast extract per liter of 50 mM NaKPO4,
pH 7.5) were harvested and stored at 
20 °C until further use.
20 °C. BtuB purity was assessed by both SDS-PAGE and
the presence of a unique NH2-terminal sequence
(NH2-QDTSPD-) corresponding to that of the mature protein
(19). Protein concentration was determined on a Varian Cary UV-visible
spectrophotometer using an extinction coefficient at 280 nm of 140,000 M1 cm1 (25).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Extraction and purification of BtuB.
Protein and LPS were detected by silver staining after separation on a
12.5% SDS-polyacrylamide gel. Lane 1, low molecular weight
protein standards; lane 2, the supernatant fraction after
the extraction of isolated outer membranes with 1% Mega-10; lane
3, pooled fractions after the initial DEAE-Sepharose; lane
4, pooled fractions from the second DEAE-Sepharose column after
buffer exchange and concentration.
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Fig. 2.
Stoichiometric levels of lipopolysaccharide
bound to BtuB after purification. LPS was detected by
carbohydrate-sensitive silver staining after separation on a 12.5%
SDS-polyacrylamide gel. Lane 1, lipopolysaccharide from
E. coli serotype 055:B5 (1.0 µg) demonstrating the
characteristic ladder-like banding pattern of LPS comprised of lipid A,
core sugars, and various numbers of O-antigen (2); lane 2, LPS from E. coli EH100 (26 pmol) comprised of only lipid A
and core sugars; lane 3, total LPS isolated from the BtuB
overexpression strain TNE012; lane 4, BtuB (26 pmol) after
final purification and desalting.
-D-octylglucoside
(Fig. 3A,
) are characteristic of proteins integral to the
bacterial outer membrane (5, 12), with a single minimum at 216 nm
(arrow, a), a cross-over point at approximately 205 nm, and
a relatively large peak of positive ellipticity centered around 195 nm.
The averaging of the secondary structure values obtained from spectral analysis by the methods of Provencher and Glockner (31) and Chang
et al. (32) indicates a predominantly -sheet secondary structure (76 ± 4%; n = 3) and a small
-helical content (15 ± 3%; n = 3).
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Fig. 3.
Analysis of secondary structure by circular
dichroism and SDS-PAGE. A, far UV CD spectra of purified
BtuB in the presence of 1% OG ( ), 1% OG/1% SDS
mixed micelles after a 15-min incubation at room temperature
(······), or 1% OG/1% SDS mixed micelles after a 2-min
incubation at 90 °C
(
···).
B, SDS-PAGE analysis of receptor conformation. Lane
1, low molecular weight protein standards; lane 2, BtuB
incubated for 2 min at 90 °C in SDS-PAGE sample buffer; lane
3, BtuB incubated for 2 min at room temperature in SDS-PAGE sample
buffer.
-barrel secondary
structure that migrates through the gel more readily than protein
denatured by a combination of SDS and heat. Similar migration patterns
are observed when BtuB is analyzed by SDS-PAGE, with the heat-treated receptor indicating a Mr value close to the true
molecular weight (Fig. 3B, lane 2); unheated samples
underestimate the true Mr value by approximately
20,000 (Fig. 3B, lane 3). The far UV CD spectrum
of unheated receptor in the presence of OG/SDS mixed micelles (Fig.
3A,······ ) demonstrates that secondary
structure is retained in the presence of SDS, directly supporting the
above interpretation of the migration patterns observed after SDS-PAGE. The incubation of BtuB at 90 °C in the presence of OG/SDS mixed micelles (Fig. 3A,
···) alters
the secondary structure. The resulting spectrum has minima at
approximately 208 and 222 nm (arrows, b and c),
which is characteristic of some -helical content, but lacks the
positive peak of large amplitude centered at 190-195 nm (37).
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Fig. 4.
Sedimentation equilibrium and sedimentation
velocity analysis of octylglucoside-solubilized BtuB. BtuB
solubilized in 15 mM NaH2PO4 and
1% OG, pH 7.5, was analyzed by sedimentation equilibrium (0.35 mg/ml)
and sedimentation velocity (0.5 mg/ml). A, equilibrium
analysis carried out for 30 h at 10,000 rpm at 20 °C with
protein absorbance monitored at 275 nm. A, radial
distribution of the protein at equilibrium. Inset, data in
A plotted as ln A versus r2, where
r and A represent radial distance in centimeters and protein
absorbance, respectively. B, distribution of S values from
sedimentation velocity measured at 50,000 rpm at 20 °C with scans
recorded at 10-min intervals.
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Fig. 5.
Effects of BtuB on the sedimentation profile
of vitamin B12. Sedimentation velocity was measured at
50,000 rpm at 20 °C with scans shown at 10-min intervals.
A, the sedimentation profile of BtuB (15 µM)
measured at 275 nm. Protein absorbance has been reduced by a factor of
4 and offset by 1 absorbance unit. B, sedimentation profile
of vitamin B12 (15 µM) measured at 358 nm
after preincubation with an equimolar concentration of BtuB.
C, sedimentation profile of vitamin B12 (30 µM) after preincubation with BtuB (15 µM).
Vitamin absorbance at 358 nm has been offset by 1.5 absorbance units.
The elevation of the baseline observed in C results from one
equivalent of free vitamin B12 that does not sediment
significantly.
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Fig. 6.
Effects of colicin E3 on the sedimentation
profile of BtuB. Sedimentation velocity analysis of BtuB
(e-h) and BtuB after preincubation with stoichiometric
levels of colicin E3 (e and h). Sedimentation
velocity was measured at 50,000 rpm at 20 °C and BtuB concentrations
of 0.5 mg/ml. Scans were recorded at 10-min intervals, and every third
scan is shown.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-helices. Circular dichroism analysis of BtuB in the presence of the
nonionic detergent octylglucoside and octylglucoside/SDS mixed micelles
indicated a predominantly -sheet secondary structure with low
-helical content, providing the first spectroscopic characterization
of the BtuB secondary structure.
-helix formation (43), suggests that this spectrum may
arise from a largely unordered species that adopts a certain degree of
-helical secondary structure upon heat denaturation in the presence
of SDS. Because unordered proteins are not represented in the
algorithms used to evaluate BtuB secondary structure content, it would
be of interest to analyze such spectra with algorithms that include
unordered proteins in the reference set (44).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Biological Sciences, 1392 Lilly Hall, Purdue University, West
Lafayette, IN 47907-1392. Tel.: 765-494-4956; Fax: 765-496-1189;
E-mail: wac{at}bilbo.bio.purdue.edu.
-D-octylglucopyranoside; PAGE, polyacrylamide gel electrophoresis; Tris, (Tris
[hydroxymethyl]aminomethane).
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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