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INTRODUCTION |
The impermeability of their cell wall renders mycobacteria
intrinsically resistant against many compounds that are toxic to other
bacteria. This is of particular medical importance in the case of
Mycobacterium tuberculosis, because it strongly limits the
number of drugs available for treatment of tuberculosis (1). The
central part of the mycobacterial cell wall is an asymmetrical bilayer
(2), consisting of an inner leaflet of very long-chain fatty acids, the
mycolic acids, and of many other unusual lipids, which form the outer
leaflet (3). This outer membrane is of exceptionally low fluidity (4)
and is estimated to be about 10 nm thick (5, 6). Pore-forming proteins
mediate the uptake of hydrophilic compounds across this outer membrane.
The existence of such porins in mycobacteria was discovered 10 years
ago (7), but only two mycobacterial porins were identified so far.
OmpATb of M. tuberculosis has low channel activity in
vitro (8) and is important for the adaptation of M. tuberculosis to low pH and for survival in macrophages and mice
(9). MspA was identified as a channel-forming protein in
chloroform/methanol extracts of Mycobacterium smegmatis
(10). Recently, it was shown that MspA is the major porin of
M. smegmatis. The permeability of the cell wall
of M. smegmatis for glucose is reduced 4-fold in an
mspA deletion mutant (11). Electron microscopy and
cross-linking experiments revealed that MspA is a tetrameric protein
forming a central pore of 10 nm length (12). This is drastically
different from the trimeric porins of Gram-negative bacteria, which
have three pores per molecule and are 4 nm long (13). These properties classify MspA as the prototype of a new family of pore proteins, since
three other porins MspB, MspC, and MspD were identified in M. smegmatis, which differ only by a few amino acids from MspA (11).
Crystal structures of outer membrane proteins from Gram-negative bacteria revealed a
-barrel as a common architectural principle (13-15). However, nothing is known about the secondary structure of
mycobacterial outer membrane proteins in general and of mycobacterial porins in particular.
It was noted earlier that MspA, which was purified by preparative gel
electrophoresis, did not dissociate in the presence of denaturing
agents such as urea or after boiling in detergents (10). Selective
extraction of functional MspA by extended heating of entire cells of
M. smegmatis in the presence of nonionic detergents to
100 °C indicated that MspA is indeed a very stable channel-forming protein (16). Since a stable pore protein would be of great value as a
detection unit in biosensors (17) and as a nanoreactor for synthesis of
nanoparticles and nanowires (18), a biochemical analysis of
purified MspA is needed to correlate the oligomer stability with its
functional activity.
In this study, we investigated the secondary structure of MspA by
circular dichroism and infrared spectroscopy. Despite the drastically
different architecture compared with porins of Gram-negative bacteria,
antiparallel
-sheets were identified as the main secondary structure
elements of MspA similar to those of other porins. We found that the
-sheet structure of purified MspA is extremely stable against
denaturation. Furthermore, tetrameric MspA withstands proteases and
very acidic and basic conditions. This biochemical analysis of a
mycobacterial porin revealed exceptional properties of a new class of
channel-forming proteins.
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EXPERIMENTAL PROCEDURES |
Chemicals and Enzymes--
Chemicals were obtained from Merck,
Roth (Karlsruhe, Germany), or Sigma (München, Germany) at the
highest purity available. The detergents SDS and
n-octylpolyoxyethylene
(octyl-POE)1 were from SERVA
(Heidelberg, Germany) and Bachem (Heidelberg, Germany), respectively.
Trypsin was from Sigma. Proteinase K was from U. S. Biochemical Corp.
Purification of Native and Recombinant MspA--
Native MspA was
extracted from M. smegmatis mc2155 using a
buffer containing 0.5% octyl-POE and purified as described (16). MspA
eluted from the anion exchange column at 0.57 M NaCl and was separated from another porin in the extracts, MspC, which eluted at
1.3 M NaCl (19). Pure MspA was finally stored in 25 mM sodium phosphate buffer (pH 7.5) containing 0.5%
octyl-POE (NaP-OPOE buffer). Porin preparations were analyzed for
purity by SDS-polyacrylamide gel electrophoresis and for
channel-forming activity by lipid bilayer measurements. Recombinant
MspA (rMspA) was overexpressed in Escherichia coli BL21 from
the expression vector pMN501, and the monomeric form of rMspA was
chromatographically purified (to be published elsewhere). Protein
concentrations were determined using bicinchoninic acid (20) and bovine
serum albumin as standards.
Circular Dichroism Spectroscopy--
CD spectra of MspA were
measured at a protein concentration of 0.26 mg/ml in NaP-OPOE buffer
using a Jasco J-715 spectropolarimeter (Japan Spectroscopic Co., Ltd.)
and a cuvette with an optical path length of 1 mm. The sample volume
was 350 µl. Buffer base lines were measured under identical
conditions and subtracted from the spectra. The raw data, obtained as
ellipticity,
, were smoothed using the standard analysis program
(Jasco). These data were transformed to mean residue molar ellipticity
[
]MRW by the equation [
]MRW =
× Mr/10,000 × n × cg × l, where
Mr denotes the molecular mass of the protein,
n is the number of residues, cg is
the concentration of the protein in g/ml, and l is the cell path in cm (from technical report No. 44, European Chirality Services; available on the World Wide Web at digilander.libero.it/ecssrl.
Attenuated Total Reflection Fourier Transform Infrared
Spectroscopy (ATR-FTIR)--
100 µg of MspA in NaP-OPOE buffer was
either dialyzed for 2 days against water or 5 mM sodium
phosphate buffer (pH 7.4) to remove the detergent. The protein remained
soluble due to the formation of ordered supramolecular assemblies (12).
Samples were dried onto a germanium crystal, which was mounted in a
self-constructed device allowing a subsequent proton-deuterium (H-D)
exchange. Spectra were recorded with a Nicolet 740 FTIR spectrometer
from 4000 to 600 cm
1 applying the ATR technique. H-D
exchange was performed by flushing the sample with
D2O-saturated nitrogen for 60 min. The kinetic of
deuteration was monitored by taking spectra every 1-5 min and judged
by shifts of the amide I and amide II bands. Final spectra were means
of 512 scans at a resolution of better than 2 cm
1. For
heat denaturation studies, MspA was suspended in 5 µl of D2O at a concentration of about 6 mg/ml. The incubation
took place in a gas-tight chamber equipped with CaF2
windows and Peltier elements, which allow the application of
temperatures from 1 °C to beyond 100 °C without loss of liquid.
Spectra were recorded every 10 °C in the range between 30 and
110 °C. The secondary structure composition was assessed using
secondary derivatives and Fourier self-deconvolution for peak
determination (21), and algorithms were implemented in the IGOR Pro 4.0 wave analysis software (WaveMetrics, Lake Oswego, OR) for constrained
band fitting and quantitative analysis.
Analysis of the Stability of MspA--
Samples of 1 µg of MspA
(concentration = 25 ng/µl) in NaP-OPOE buffer were incubated for
15 min at temperatures ranging from 20 to 100 °C, followed by rapid
cooling to 4 °C. Temperatures were increased in 10 °C steps using
a thermal cycler (Mastercycler gradient; Eppendorf), which maintained
temperatures within a range of ±0.2 °C. The upper temperature limit
was given as 99 °C by the manufacturer and approximated 100 °C in
Figs. 1 and 2. Samples of 1 µg of MspA (concentration = 25 ng/µl) in NaP-OPOE buffer were supplemented to final concentrations
of 2% SDS or 7.6 M urea and incubated as described above.
Urea was removed by dialysis against 400 ml of NaP-OPOE buffer for
12 h at room temperature to avoid band broadening in
polyacrylamide gels. Electrophoretic analysis of urea-containing MspA
samples without prior dialysis in 6 M urea gels yielded the
same result for the stability of MspA, indicating that dialysis did not
lead to a detectable refolding of denatured MspA. Samples containing
800 ng of MspA were loaded on denaturing polyacrylamide gels and
stained as indicated. The stability of purified MspA at different pH
values was analyzed using samples of 500 ng of MspA in NaP-OPOE buffer,
which were diluted 10-fold in the following solutions to a final
concentration of 25 ng/µl and incubated for 1 h at 20 °C. The
pH values were adjusted as described (22) by 1, 0.1, and 0.01 M HCl to pH 0, 1, and 2, respectively; 20 mM
glycine/HCl to pH 3; 20 mM sodium citrate/citric acid to pH
4, 5, and 6; 20 mM HEPES/NaOH to pH 7 and 8; 20 mM glycine/NaOH to pH 9-11; and 0.01, 0.1, and 1 M NaOH to pH 12, 13, and 14, respectively. The stability of
MspA in crude extracts at different pH values was analyzed using
samples of 2 µl of crude extract of M. smegmatis
mc2155 in POP05 buffer (100 mM
Na2HPO4/NaH2PO4, 0.1 mM EDTA, 150 mM NaCl, 0.5% (w/v)
octyl-POE, pH 6.5), (16). The samples were mixed with 18 µl of
the same buffers as for pure MspA and incubated for 1 h at
20 °C. The proteins in these samples were analyzed by gel
electrophoresis, and the channel activity was determined by lipid
bilayer measurements as described below.
Gel Electrophoresis and Gel Analysis--
Protein samples were
analyzed in denaturing polyacrylamide gels, which were stained with
silver (16). All samples were mixed with loading buffer (140 mM Tris, 4% SDS, 30% glycerol, 0.1% bromphenol blue, pH
7.5) and incubated for 10 min at room temperature before loading on a
denaturing 10% polyacrylamide gel (23) if not stated otherwise. For
quantitative analysis of the stability experiments, the gels were
scanned, and relative protein amounts were determined from the gel
images using the densitometry plugin of the program NIH Image 1.62 (available on the World Wide Web at rsb.info.nih.gov/nih-image/). The
brightness and the global gradation curve of the images were adjusted
to reduce the background of the figures. No parts of the gels were
changed individually. The midpoint of thermal transition of MspA
(Td50) was defined as the temperature at which
the amount of tetrameric MspA was reduced by 50% as compared with that
at 20 °C.
Lipid Bilayer Experiments--
The channel activity of selected
samples was measured in lipid bilayer experiments as described (16). In
all lipid bilayer experiments, the lipid bilayer was made from
diphytanoyl-phosphatidylcholine, and the aqueous phase contained 1 M KCl and 10 mM 2-MES buffer (pH 6.0). The
temperature was kept at 20 °C using a thermostat. MspA extracts were
incubated at different pH values and subsequently diluted 100-fold with
the extraction buffer (16) for the pH stability experiments. The MspA
concentration in the cuvette was estimated to 25 pg/ml. At least 60 MspA pores in four membranes were recorded in each single channel experiment.
To determine the influence of heat, SDS, or urea on the pore forming
activity of MspA, samples of 1 µg of MspA (concentration = 25 ng/µl) were incubated at different temperatures for 15 min in
NaP-OPOE buffer without additional agents and in the presence of 2%
SDS and 7.6 M urea. After incubation, the samples were
diluted with NaP-OPOE buffer to a final concentration of 15.7 pg/µl
MspA. Then 78.5 pg of MspA from each sample was added to the KCl
solution bathing the black lipid membranes. The final concentration of MspA in the bilayer cuvette was 7.8 pg/ml.
Fluorescence Measurements--
Fluorescence measurements were
carried out on a Fluorolog-1680 double spectrometer (SPEX Industries,
Inc.) using an excitation and emission bandpass of 3.6 nm. An internal
rhodamine B standard (Eastman Kodak Co.) was used to correct intensity
fluctuations of the xenon arc lamp. MspA samples (13 µg,
concentration = 10 µg/ml) were incubated in the presence of 7.6 M urea for 15 min at temperatures increasing in 10 °C
steps from 30 to 100 °C. The samples were cooled to 22 °C, and
this temperature was maintained throughout the measurement.
Fluorescence of MspA was excited at 280 nm, and emission was recorded
from 282 to 492 nm. The midpoint of thermal transition of MspA
(Td50) in the presence of urea was defined as
the temperature where the maximal fluorescence intensity of MspA was
50% of that at 30 °C.
Proteolysis--
Tetrameric MspA (15 µg, concentration = 0.6 µg/µl) was treated with trypsin and proteinase K in 25 mM NaP-OPOE buffer. Monomeric rMspA (15 µg,
concentration = 0.6 µg/µl) was incubated with the same
proteases in 25 mM sodium phosphate buffer (pH 7.5), which did not contain any detergents. The enzyme/protein ratio was 1:25 and
1:5 (w/w). All protease reactions were performed at 37 °C for 1.5 or
12 h. As a control, MspA was incubated under the same conditions,
but the proteases were omitted. Digests were stopped by adding
4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC; Roche
Diagnostics) to a final concentration of 2 mM, which irreversibly blocks serine proteases such as trypsin and proteinase K. Samples were then mixed with loading buffer and loaded on a denaturing
10% polyacrylamide gel. The gel was blotted onto a nitrocellulose
membrane, and MspA was detected using the polyclonal antiserum pAK#813
(10), a secondary anti-rabbit antibody (Sigma), and the ECLplus kit
(Amersham Biosciences).
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RESULTS |
Secondary Structure of MspA--
The major porin of M. smegmatis, MspA, was selectively extracted by boiling entire cells
for 30 min in a buffer containing 0.5% octyl-POE, precipitated with
acetone to remove lipids, and subsequently purified by anion exchange
and gel filtration chromatography to remove contaminating small
compounds and proteins as described earlier (16). This approach is
currently the only method that yields apparently pure MspA in milligram
quantities. Since the tetrameric MspA pore has a projection structure
drastically different from that of other porins (12) and is the first
outer membrane protein isolated from mycobacteria that is amenable to
structural investigations, the secondary structure of purified MspA was
analyzed. Circular dichroism spectra showed a large positive mean molar ellipticity below 205 nm, a crossover point at 206 nm, at which the
ellipticity equaled zero, and a single minimum at 215 nm (Fig. 1A). These features are
typical of
-sheet structures (24) and were similar to those obtained
for
-barrel proteins such as the porins of Gram-negative bacteria
(22).

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Fig. 1.
Secondary structure of MspA.
A, CD spectrum. The ellipticity of MspA (260 µg/ml) was
recorded at 22 °C in NaP-OPOE buffer (25 mM phosphate,
pH 7.5, containing 0.5% n-octylpolyethylene oxide) and
transformed into the mean residue molar ellipticity
[ ]MRW (solid line). The zero
line is indicated by dots. B, amide I region of
infrared absorbance spectra (ATR-FTIR) of MspA and of porin Omp32 from
D. acidovorans. MspA (100 µg dissolved in 25 mM phosphate buffer and 1% octyl-POE) was dialyzed against
water, and the sample was dried onto a germanium crystal. The spectra
of MspA prior to (long dashes) and after
(black solid line) H-D exchange are
plotted together with the spectrum of porin Omp32 without H-D exchange
(gray solid line). The peak at 1630 cm 1 is indicative of -strands. The more prominent peak
of the Omp32 spectrum denotes a higher relative -structure content
compared with MspA. The spectra were normalized to a maximum absorption
of 1.
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IR spectroscopy is known to yield a particularly reliable estimate of
the
-sheet content of proteins (25). Therefore, IR absorbance
spectra of MspA were analyzed for the spectral components of the amide
I band region (1700-1600 cm
1), which is indicative for
the secondary structure of proteins (26), and compared with that of
porin Omp32 from Delftia (Comamonas) acidovorans. The latter has a
-sheet content of 61% as
derived from its atomic structure (27). IR spectral analysis of Omp32 yielded a
-sheet content of 59%, in excellent agreement with the
structural data. IR spectra of MspA showed a peak around 1632 cm
1 (Fig. 1B), which is typical for proteins
with predominant
-structure. Fourier self-deconvolution for band
positioning and subsequent fit of corresponding absorption bands to
MspA spectra taken after hydrogen-deuterium (H-D) exchange followed by
quantitative analysis revealed a secondary structure composition of
48-52%
-sheet, 10-15%
-helix, and remaining structure
composed of
-turns and random folds. The lower relative content of
-structure in MspA compared with that of porin Omp32 is consistent
with a less prominent peak at 1632 cm
1 (Fig.
1B). The small peak at 1692 cm
1 in the IR
spectrum is indicative of antiparallel
-sheets (26) and very similar
to spectra of porins from Gram-negative bacteria. Secondary structure
predictions based on the MspA sequence and calculated using seven
different algorithms yielded a consensus assessment containing about
45%
-structure and 10%
-helix, in agreement with the IR
spectra. Taken together, IR and CD spectroscopy consistently indicated
-sheets as the main secondary structure elements of MspA.
Structural and Functional Stability of MspA in the Presence of
SDS--
Since earlier experiments indicated a very high thermal
stability of MspA (10, 16), we wanted to examine whether the resistance of the MspA tetramer against dissociation correlated with the stability
of its secondary structure and its channel activity. Our first aim was
to precisely determine the thermal stability of MspA in the nonionic
detergent octyl-POE, which was used for purification. MspA (25 ng/µl)
was incubated for 15 min at different temperatures and
electrophoretically analyzed. Fig. 2
shows that MspA tetramers with an apparent molecular mass of 100 kDa
were detected after incubation at 100 °C, illustrating that even
boiling is not sufficient to completely denature MspA. Thus, the
temperature required for complete conversion of MspA to monomers is at
least 25 °C higher than that of trimer dissociation of the E. coli porin OmpF, which dissociated completely at about 75 °C
and is considered to be an extremely stable protein (28, 29).
Quantitative image analysis of the gels revealed that about 50% of the
MspA tetramers were dissociated at 92 °C (Table
I). It should be noted that we did
not detect the MspA monomer in octyl-POE extracts after heating
M. smegmatis cells to 100 °C for 30 min (16), indicating that a stabilizing factor was lost during the subsequent purification steps. The addition of SDS to a final concentration of 2% to the purified MspA tetramer did not significantly reduce its thermal stability (Table I) in contrast to OmpF, whose midpoint of thermal transition was reduced to 59 °C in the presence of SDS (28).

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Fig. 2.
Thermal stability of the MspA tetramer.
Samples of 1 µg of purified MspA (25 ng/µl) were incubated for 15 min at different temperatures, which are indicated above the
lanes in degrees centigrade. 800 ng of MspA of each sample
was analyzed using a silver-stained denaturing 10% polyacrylamide gel.
Lane M, molecular mass marker; lane
C, untreated MspA as a control (800 ng). The gel was scanned
and quantitatively analyzed using the program NIH Image 1.62. It should
be noted that the MspA monomer was stained 1.5-fold more efficiently
with silver than the tetramer.
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Table I
Thermal stability of MspA in the presence of denaturing agents
Td1, Td50, and
Td100 were defined as temperatures at which the band
of monomeric MspA started to appear, the intensity of the band of
tetrameric MspA was reduced to 50%, and no tetrameric MspA was
detected anymore, respectively. These temperatures were determined by
quantitative image analysis, using the software NIH Image 1.62, of
silver-stained, denaturing polyacrylamide gels (see Figs. 2 and
5A). The temperature at which the -sheet content (Fig. 3)
and the intensity of the fluorescence maximum (Fig. 5B) were
decreased to 50% was defined as TM and is
equivalent to the temperature Td50 derived from gel
electrophoresis. Due to experimental limitations, IR (infrared) spectra
could not be recorded at temperatures above 110 °C, and no IR
spectra of fully denatured MspA were measured. Therefore, the formal
-sheet content of denatured MspA was estimated to 5%, similar to
that of other proteins (H. Engelhardt, unpublished data). The
denaturation curve of MspA without SDS was extrapolated to determine
TM. ND, not determined.
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Next, we examined whether the secondary structure of the MspA tetramer
was affected at temperatures close to 100 °C, where gel
electrophoresis showed that the MspA tetramer resisted dissociation into monomers even in the presence of denaturing agents. An MspA sample
dissolved in D2O was incubated for 15-20 min in a special thermochamber at temperatures increasing in steps of 10 °C and in
steps of 3 °C above 100 °C. IR spectra were recorded at each selected temperature (Fig.
3A). At 30 °C, the maximum
of the amide I band in the IR spectrum appeared at 1634 cm
1 and was constant up to ~80 °C. The peak position
of the amide I band increased slightly up to ~100 °C and shifted
drastically to higher wave numbers above ~100 °C, indicating the
temperature-induced unfolding of MspA (Fig. 3, A and
B).

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Fig. 3.
Thermal stability of the secondary structure
of MspA. A, amide I region of infrared spectra
of MspA. The protein was dialyzed to remove the detergent, dried, and
dissolved in D2O at a concentration of about 6 mg/ml. A
sample of 5 µl was continuously heated in a gas-tight chamber for
15-20 min at the indicated temperatures. B, plot of the
peak position of the amide I band (circles) and of the
relative content of -structure (triangles). Samples were
dissolved in D2O (black curves) or
D2O including 2% SDS (red curves).
The cumulative incubation time for the sample without SDS is displayed.
The respective time for the sample containing SDS was close to the
given values.
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The amide I band region of the deconvoluted spectra was analyzed for
fractional intensities of the secondary structure elements by curve
fitting of spectral components as described above. The changes of the
-sheet content of MspA with increasing temperatures are depicted in
Fig. 3B. At 30 °C, curve fitting yielded a
-sheet content of 45%, in good agreement with the ATR-FTIR spectrum shown above (Fig. 1B). The
-strand content decreased slightly
to a relative amount of 40% at 100 °C but dropped sharply at
temperatures above ~100 °C, indicating denaturation of the
protein. Thus, both the peak position of the amide I band and the
calculated
-sheet content consistently indicated unfolding of MspA
at temperatures above ~100 °C.
The addition of 2% SDS to MspA did not lead to visible differences in
the IR spectra as compared with those depicted in Fig. 3A.
Quantitative analysis revealed minor changes of the peak position of
the amide I band at temperatures below ~100 °C and drastic changes
at temperatures above ~100 °C (Fig. 3B). The
-sheet
content of MspA in the presence of 2% SDS decreased to 40% at
50 °C, remained constant up to ~80 °C, and dropped sharply to
almost 20% at temperatures above ~100 °C. These results
demonstrated that SDS had a significant but minor effect on the thermal
stability of the overall secondary structure of MspA.
Lipid bilayer experiments were used to analyze whether the activity of
MspA as a channel protein correlated with its apparent structural
stability. First, we determined the minimal concentration of MspA,
which produced a rather low frequency of reconstitution events in the
range of 10 pores/min. 3-7 pores/min reconstituted into
diphytanoyl-phosphatidylcholine membranes after the addition of MspA at
a concentration of 7.8 pg of MspA/ml of KCl in the bilayer cuvette
(Fig. 4A). Then samples of 1 µg of MspA were incubated for 15 min at 50, 90, and 100 °C. No
significant change in the channel activity of MspA was observed at
temperatures up to 90 °C. Increasing the temperature to 100 °C
reduced the channel activity about 5-fold, but a residual activity of
0.8 ± 0.7 pores/min was still detected (Fig. 4C). This
result is in good agreement with the existence of MspA tetramers and a
slightly reduced
-sheet content of about 40% at 100 °C observed
by gel electrophoresis and IR spectroscopy, respectively. Surprisingly,
the addition of 2% SDS reduced the channel activity of MspA
significantly even at 20 °C. A 7-fold reduction of the channel
activity was already observed at 50 °C and higher temperatures,
although the MspA tetramer was stable at this temperature (Fig. 2), and
the
-sheet content was not reduced (Fig. 3).

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Fig. 4.
Influence of denaturing conditions on the
single channel activity of MspA. In all lipid bilayer experiments,
the conductance of a diphytanoyl phosphatidylcholine membrane in the
presence of 7.8 pg/ml purified MspA was recorded. MspA was incubated
for 15 min in NaP-OPOE buffer at different temperatures, without
additional agents and in the presence of 2% SDS and 7.6 M
urea before activity measurements. MspA was added to both sides of the
membrane. A, single channel activity of MspA incubated at
20 °C without additional agents. Data were collected from four
different membranes. Seven out of 147 reconstitution events are shown.
The data were collected from four different membranes. Mainly,
conductance steps of about 4.6 nanosiemens were observed. B,
single channel activity of MspA after incubation in the presence of 2%
SDS at 50 °C. The data were collected from three different
membranes. Due to the low activity of MspA under those conditions, only
10 reconstitution events with conductance steps of 4.6 nS were
recorded. C, analysis of the single channel activity of MspA
in dependence on the temperature and the presence of denaturing agents.
The frequency of pore insertions measured in lipid bilayer experiments
is depicted. White, hatched, and gray
bars denote the channel activity of MspA without additional agents,
with 2% SDS, and with 7.6 M urea, respectively. In
experiments, when the channel activity of MspA was drastically reduced
(100 °C without agents, 50 °C and 100 °C with 2% SDS, and
50 °C with 7.6 M urea), between 10 and 30 single channel
events from at least three different membranes were analyzed. In all
other experiments, at least 100 reconstitution events from at least
three different membranes were analyzed.
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Structural and Functional Stability of MspA in the Presence of
Urea--
Urea-dependent denaturation experiments were
used to analyze whether the thermal stability of MspA also extended to
other potent denaturing agents, which interact differently with protein surfaces than detergents (30). Gel electrophoresis revealed that the
MspA tetramer was completely stable in a solution containing 7.6 M urea at 20 °C (Fig.
5A). Half of the MspA
tetramers were dissociated at 89 °C. Thus, the transition
temperature Td50 of MspA did not change
regardless of whether 2% SDS or 7.6 M urea were present
(Table I). However, the MspA monomer was visible at ~70 °C, which
was ~10 °C below the MspA control sample, and dissociation of the
MspA tetramer was complete at 100 °C (Fig. 5A) in
contrast to the MspA control or after the addition of SDS, suggesting
that 7.6 M urea slightly reduced the thermal stability of
MspA tetramers.

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Fig. 5.
Stability of MspA in urea at different
temperatures. A, samples of 1 µg of purified MspA (25 ng/µl) were incubated for 15 min at different temperatures between 20 and 100 °C in the presence of 7.6 M urea. Urea was
removed by dialysis, and 800 ng of MspA of each sample was analyzed
using a silver-stained denaturing 10% polyacrylamide gel. The
incubation temperatures are indicated above the
lanes. Lane M, molecular mass marker;
lane C, untreated MspA as a control (800 ng). The
gel was scanned and quantitatively analyzed using the program NIH Image
1.62. It should be noted that the MspA monomer stained ~1.7-fold more
efficiently with silver than the tetramer. B, fluorescence
spectra of MspA after incubation in urea at different temperatures.
MspA at a concentration of 10 µg/ml was incubated for 15 min at
30 °C (solid line), 70 °C (long
dashes), and 100 °C (medium dashes)
in the presence of 7.6 M urea. The excitation wavelength
was 280 nm, and the emission spectra were recorded at 22 °C.
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MspA at a concentration of 10 µg/ml showed a large fluorescence
emission peak at 347 nm after excitation at 280 nm (Fig.
5B). Since detection of MspA fluorescence was more sensitive
than detection of the CD and IR signal, fluorescence was used to
analyze the thermal denaturation of MspA in the presence of urea in
more detail. The maximal fluorescence of MspA slightly decreased after
increasing the temperature in 10 °C steps up to 100 °C for 15 min
(Fig. 5B). At 100 °C, the fluorescence maximum of MspA
was shifted to 361 nm (Fig. 5B), indicating that the four
tryptophans of MspA underwent a transition from a more hydrophobic to a
solvent-exposed state (31). A quantitative data analysis revealed a
midpoint of transition at 84 °C in good agreement with the
electrophoretic analysis (Table I). These results suggested a major
structural change of MspA consistent with dissociation and unfolding
under these conditions. The channel activity of MspA was not
significantly affected by 7.6 M urea at 20 °C in lipid
bilayer experiments. However, raising the temperature to 50 °C
reduced the channel activity of MspA ~30-fold (Fig. 4C),
although the tetramer was stable (Fig. 5A), and the
fluorescence spectrum did not show any major changes of the environment
of the four tryptophans at this temperature (Fig. 5B). Thus,
both denaturing agents, SDS and urea, strongly reduced either the
channel structure or the reconstitution efficiency of MspA into
artificial bilayers without major structural changes detectable by IR
and fluorescence spectroscopy or gel electrophoresis.
pH-dependent Structural and Functional Stability of
MspA--
To examine the stability of MspA under acidic and basic
conditions, samples of 500 ng of purified MspA (25 ng/µl) were
incubated at pH values ranging from 0 to 14 for 1 h at 20 °C.
Surprisingly, gel electrophoresis showed that MspA oligomers were
stable at any pH (Fig. 6A). At
very low pH values (0-2), faint bands of the MspA monomer were
visible, and formation of SDS-stable high molecular mass aggregates of
MspA was induced at pH 2 and below (Fig. 6A). Since the
purified MspA tetramer was less stable against thermal denaturation
than MspA in octyl-POE extracts of M. smegmatis, the pH
stability of MspA before and after purification was compared. MspA is
clearly the most dominant protein in octyl-POE extracts (Fig.
6B), since the extraction procedure is rather selective for
porins (16). No monomer was detectable after incubation of MspA at any
pH from 0 to 14 for 1 h at 20 °C (Fig. 6B), although the monomer was stained 1.5-fold more efficiently than the oligomer (compare Fig. 2). In all samples, pores reconstituted rapidly into
planar lipid membranes at an estimated protein concentration of ~25
pg/ml (not shown). Even more surprisingly, the average reconstitution
rate was ~6 ± 3 pores/min at pH 0, 1, 7, and 14. It should be
noted that the current noise of the lipid bilayer after reconstitution
of MspA channels incubated at pH 0 was significantly increased compared
with measurements at other pH values. Nevertheless, in all experiments,
the single channel conductance of more than 50% of the reconstituted
pores was 4.5 nS, in agreement with earlier results (10). These results
demonstrated that MspA in octyl-POE extracts was resistant against
denaturation at any pH from 0 to 14. Formation of SDS-stable aggregates
occurred at pH 0 and 1 (Fig. 6B). Since we observed neither
a shift to larger single channel conductances nor a drop in the channel
activity of these aggregates (data not shown), we concluded that the
structure and the reconstitution efficiency of the MspA pore were not
irreversibly affected by aggregation. Formation of SDS-stable and
functional aggregates was also observed after extraction of MspA from
M. smegmatis with a mixture of chloroform and methanol (10)
and appears to be an inherent property of MspA (12). Furthermore, these
results showed that MspA in octyl-POE extracts was more stable than
purified MspA. Since the thermal stability of purified MspA was also
significantly lower than that in extracts (see above), it is concluded
that a stabilizing compound was lost during purification of MspA. Most
likely, lipids were partially removed from MspA by the precipitation
steps using acetone (16), which might explain the reduced stability of
purified MspA against denaturation. Nevertheless, purified MspA is
considerably more stable against pH changes than the E. coli
porin OmpF, which irreversibly loses its trimeric structure at pH
values lower than 4 and above 12.4 (22, 28).

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Fig. 6.
Stability of MspA against extreme pH
values. A, 500 ng of MspA was incubated for 1 h at
different pH values (indicated above the lanes)
and analyzed using a silver-stained denaturing 10% polyacrylamide gel.
After loading, the pH of all samples was above 4.6. B, 2 µl of crude extract of M. smegmatis mc2155
(16) containing ~50 ng of MspA was incubated for 1 h at
different pH values and analyzed using a silver-stained denaturing 10%
polyacrylamide gel.
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Protease Resistance of MspA--
No degradation products of
purified MspA were observed after proteolysis with trypsin or
proteinase K for 30 min at 37 °C (data not shown). Increasing the
enzyme/MspA ratio from 1:20 to 1:5 (w/w) and extending the incubation
time to 12 h did not lead to detectable degradation of tetrameric
MspA with both proteases in a Western blot (Fig.
7, lanes 4 and
5). In contrast, monomeric rMspA is susceptible to both
proteases and is completely degraded under these conditions (Fig. 7,
lanes 7 and 8). It should be noted that tetrameric rMspA, which apparently self-assembled in E. coli (Fig. 7, lane 6) and had channel
forming activity as observed earlier (10), was partially resistant to
trypsin under these conditions (Fig. 7, lane 7).
The apparent partial degradation of tetrameric rMspA could be due to a
relative excess of trypsin compared with the experiments with native
MspA. Alternatively, this result may indicate that rMspA tetramer
formed in E. coli is not identical with MspA isolated from
M. smegmatis.

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Fig. 7.
Protease resistance of MspA. 15 µg of
both purified MspA from M. smegmatis and recombinant MspA
from E. coli (rMspA) were incubated with 3 µg of trypsin
and proteinase K in a 25 mM phosphate buffer (pH 7.5) for
12 h at 37 °C. The protease reaction was stopped by adding an
inhibitor. The samples were separated on a denaturing 10%
polyacrylamide gel, blotted on a nitrocellulose membrane, and detected
with rabbit antiserum to purified MspA (pAK MspA#813). Lane
M, molecular mass marker; lane 1,
trypsin (20 ng); lane 2, proteinase K (20 ng);
lane 3, MspA (5 ng); lane
4, MspA (5 ng) plus trypsin; lane 5,
MspA (5 ng) plus proteinase K; lane 6, rMspA (40 ng); lane 7, rMspA (40 ng) plus trypsin;
lane 8, rMspA (40 ng) plus proteinase K.
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DISCUSSION |
The
-Strands of MspA Are Organized in an Extremely Stable
Domain--
The channel structure of the major porin MspA from
M. smegmatis is drastically different from that of all other
known porins. Four monomers build one central pore in contrast to the
trimeric porins of Gram-negative bacteria, which possess one pore per
monomer (12). Furthermore, MspA consists only of 184 amino acids
compared with ~340 amino acids of the general porins of Gram-negative
bacteria but is almost twice as long as the latter. Despite these
differences, secondary structure analysis by CD and IR spectroscopy
identified
-sheets as the main secondary structure elements of MspA.
Quantitative analysis of the IR data revealed that 45-50% of MspA is
folded in antiparallel
-sheets. The apparent decrease of
-sheet
content of MspA at temperatures above 50 °C and in the presence of
2% SDS might be explained by disintegration of MspA micelles, which are formed in the absence of detergent at low temperature (12), because
aggregation is known to contribute to the amide I peak in IR spectra of
proteins (32, 33). Taking this contribution into account, the real
-sheet content of MspA should rather be in the range of 40-45%,
corresponding to ~75-85 amino acids in
-strands. This is
significantly less compared with the general porins of Gram-negative
bacteria, where up to 60% of the amino acids build a 16-stranded
-barrel (34).
The drastic loss of
-structure above 100 °C within a temperature
range of 10-15 °C indicates that the
-strands are not
distributed over several small and distinct
-sheets but form a
larger coherent domain in the MspA monomer. For this
-sheet domain,
a transition temperature of 112 °C was determined by IR
spectroscopy. Since the thermal stability of proteins and the length of
-sheets are clearly correlated (35), this very high transition
temperature probably reflects the length of the
-strands in the MspA
monomer, which assemble to a 10-nm-long channel (12) in contrast to the porins of Gram-negative bacteria, which build 4-nm-long pores (34). Our
assumption is in excellent agreement with the observation that the
-barrel of OmpA and related
8 outer membrane proteins, which
possess particularly long
-strands (up to 20 amino acids), are not
completely denatured in SDS solutions at 100 °C (36). Hydrophobic
interactions between amino acid residues in the MspA monomer appear to
contribute little to the
-sheet stability, because the addition of
SDS lowered the transition temperature only to about 108 °C.
The Interactions between the MspA Subunits Are Different from Those
in the Porins of Gram-negative Bacteria--
The exceptional thermal
stability of MspA also applies to the functional tetramer. Significant
dissociation of the MspA complex and concomitant loss of pore formation
in conductance measurements was only observed at temperatures above
90 °C, which implies rather stable intersubunit contacts. A number
of observations illustrate that these interactions differ from those in
trimeric porins. (i) In the porins of Gram-negative bacteria, three
individual, about 4-nm-long
-barrels are in contact via areas being
created by the flat side of adjacent
-sheets (34). The situation in MspA tetramers is clearly different. The stability of the
-sheet domain in the MspA monomer suggests that the subunits interact with the
edges of their
-sheets to create a central channel with ~2.5 nm in
diameter (12). This also excludes a tertiary structure similar to that
of TolC, where three monomers are intertwined to form one central
channel (14). (ii) The intersubunit contact in trimeric porins such as
OmpF from E. coli or Omp32 from D. acidovorans
and others is mainly stabilized by hydrophobic interactions and salt
bridges, as can be derived from their atomic structures (27, 29).
Disruption of the hydrophobic interactions by SDS consequently reduced
the denaturation temperature of OmpF by 15-20 °C compared with the
nonionic and less aggressive detergent octyl glucoside (28). By
contrast, dissociation of the MspA tetramer was not increased upon
treatment with SDS (Table I). (iii) In OmpF, two glutamate-arginine
salt bridges are important for trimer stability. Uncharging one
glutamate by mutation (29) or adjusting the pH value below 4 (28) had a
significant effect on the stability of the trimer. In contrast to this
observation, MspA tetramers were essentially stable from pH 0 to 14, clearly illustrating that salt bridges do not stabilize the subunit
interactions significantly. In summary, regardless of whether we look
at dissociation of the MspA tetramer with a TM
of 92 °C or at unfolding of the
-domain with an
TM of 112 °C, MspA is, to our knowledge, the
most stable channel protein known to date, exceeding even the
remarkable stability of the general porins of Gram-negative bacteria
with transition temperatures of ~70 °C (29). The trimeric
maltose-specific porin LamB of E. coli also withstands
boiling for 30 min, but an intrasubunit disulfide bond plays a major
role in stabilizing LamB (37), in contrast to MspA and all other
general porins, which do not contain cysteines.
The
-Sheets of MspA Probably Form a
-Barrel Channel--
The
stability experiments and the structural analysis of MspA clearly
showed differences to the trimeric porins. This led us to propose that
the
-domain of MspA is part of one channel-forming
-barrel in the
functional MspA tetramer analogous to the structures of multicomponent
barrels found in
-hemolysin and aerolysin (38, 39). This model is
based on the following observations. (i)
-Barrel proteins are known
for their remarkable stability. For instance, the
-barrels of FhuA
of E. coli (22
-strands) or Omp21 from D. acidovorans (8
-strands) are stable up to 75 °C and
>80 °C (40, 41). It is interesting to note in this regard that the
stability of the
-barrel against denaturation is not coupled to
localization of those proteins in the outer membrane, since also
soluble
-barrel proteins such as the lipocalins or green fluorescent
protein show very high transition temperatures of 61 and 82 °C,
respectively (42, 43). That the
-sheet within the MspA monomers is
more stable (TM = 112 °C) than the
-strand interactions between adjacent subunits (TM = 92 °C) may be explained by, for example, fewer hydrogen bonds in
shorter contact regions. (ii) MspA was essentially stable at any pH
from 0 to 14, indicating that ionizable groups do not appear to play a
major role in subunit interactions (see above). This suggests
hydrogen bonds as the major interaction force between the MspA subunits
and favors a coherent
-barrel domain, which is predominantly held
together by hydrogen bonds (44). This fact is also reflected in the pH stability of other
-barrel proteins such as rusticyanin (45). (iii)
The relatively small thickness of the channel wall of MspA as seen in
the electron micrographs would rather fit a
-barrel structure than a
structure composed of more voluminous polypeptide foldings (12). The
expected barrel height of 6 nm or more, forming the putative
membrane-spanning portion of the channel, is in reasonable agreement
with the assumption of relatively long
-strands. (iv) Proteins
located in the inner membrane are always composed of a bundle of
hydrophobic
-helices, whereas the
-barrel was exclusively found
in outer membrane proteins (13). This was explained by the observation
that hydrophobic
-helices would prevent the polypeptide release from
the inner membrane and stop the transfer to the outer membrane (46).
This physical principle should also apply to the Gram-positive
mycobacteria, which have an outer membrane in addition to the
cytoplasma membrane (2). In conclusion, organization of the
-sheets
of the four MspA subunits in a
-barrel is the easiest model to
explain all experimental observations, although the localization and
the function of the
-domain cannot be deduced directly from our data.
The Channel Function of MspA but Not the Subunit Association Is
Affected by Denaturing Agents at Elevated Temperatures--
In
apparent contrast to the extraordinary high structural stability of
MspA, its channel activity after incubation in 2% SDS and 7.6 M urea at 50 °C was reduced 13- and 30-fold,
respectively, compared with MspA in octyl-POE at 50 °C in lipid
bilayer experiments. In principle, this could be due to either a switch
of MspA to a closed channel conformation or to a reduced reconstitution
efficiency. Voltage- and pH-dependent conformational
changes of extracellular loops leading to channel closure were
demonstrated for the general porin OmpF (47) and for the
maltose-specific porin LamB (48). Since substantial extracellular
domains were observed in electron microscopy pictures of MspA in the
cell wall of M. smegmatis (12), it seems more likely that
the urea- or SDS-induced loss of channel activity reflects structural
alterations of MspA rather than a reduced reconstitution efficiency of
MspA in lipid bilayer experiments. However, further experiments are
needed to distinguish between both possibilities.
MspA Is Protected against Denaturation by an Unknown
Compound--
This study showed that purification of MspA reduced its
stability against both thermal and acid denaturation (e.g.
the MspA monomer was detectable after incubation of purified MspA at
80 °C for 15 min (Fig. 2) in contrast to MspA in unpurified
detergent extracts of M. smegmatis (16)). Since the
detergent extracts contained only minor amounts of proteins other than
MspA, it seems likely that associated lipids increased the stability of
MspA further and were removed by acetone precipitation in the course of
purification (16). This finding is similar to porins of Gram-negative bacteria, which are tightly bound by lipopolysaccharide (49).