The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable beta -Sheet Domain*

Christian HeinzDagger , Harald Engelhardt§, and Michael NiederweisDagger

From the Dagger  Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany and § Max-Planck-Institut für Biochemie, Abteilung Molekulare Strukturbiologie, Am Klopferspitz 18a, D-82152 Martinsried, Germany

Received for publication, December 3, 2002, and in revised form, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel beta -strands organized in a coherent domain. Heating to 92 and 112 °C was required to dissociate the MspA tetramer and to unfold the beta -sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming beta -barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 M urea at 50 °C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the beta -sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are beta -sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -sheets were identified as the main secondary structure elements of MspA similar to those of other porins. We found that the beta -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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, Theta , were smoothed using the standard analysis program (Jasco). These data were transformed to mean residue molar ellipticity [Theta ]MRW by the equation [Theta ]MRW = Theta  × 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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -sheet structures (24) and were similar to those obtained for beta -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 [Theta ]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 beta -strands. The more prominent peak of the Omp32 spectrum denotes a higher relative beta -structure content compared with MspA. The spectra were normalized to a maximum absorption of 1.

IR spectroscopy is known to yield a particularly reliable estimate of the beta -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 beta -sheet content of 61% as derived from its atomic structure (27). IR spectral analysis of Omp32 yielded a beta -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 beta -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% beta -sheet, 10-15% alpha -helix, and remaining structure composed of beta -turns and random folds. The lower relative content of beta -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 beta -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% beta -structure and 10% alpha -helix, in agreement with the IR spectra. Taken together, IR and CD spectroscopy consistently indicated beta -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 beta -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 beta -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.

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 beta -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.

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 beta -sheet content of MspA with increasing temperatures are depicted in Fig. 3B. At 30 °C, curve fitting yielded a beta -sheet content of 45%, in good agreement with the ATR-FTIR spectrum shown above (Fig. 1B). The beta -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 beta -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 beta -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 beta -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 beta -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.

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.

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The beta -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 beta -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 beta -sheets. The apparent decrease of beta -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 beta -sheet content of MspA should rather be in the range of 40-45%, corresponding to ~75-85 amino acids in beta -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 beta -barrel (34).

The drastic loss of beta -structure above 100 °C within a temperature range of 10-15 °C indicates that the beta -strands are not distributed over several small and distinct beta -sheets but form a larger coherent domain in the MspA monomer. For this beta -sheet domain, a transition temperature of 112 °C was determined by IR spectroscopy. Since the thermal stability of proteins and the length of beta -sheets are clearly correlated (35), this very high transition temperature probably reflects the length of the beta -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 beta -barrel of OmpA and related beta 8 outer membrane proteins, which possess particularly long beta -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 beta -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 beta -barrels are in contact via areas being created by the flat side of adjacent beta -sheets (34). The situation in MspA tetramers is clearly different. The stability of the beta -sheet domain in the MspA monomer suggests that the subunits interact with the edges of their beta -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 beta -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 beta -Sheets of MspA Probably Form a beta -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 beta -domain of MspA is part of one channel-forming beta -barrel in the functional MspA tetramer analogous to the structures of multicomponent barrels found in alpha -hemolysin and aerolysin (38, 39). This model is based on the following observations. (i) beta -Barrel proteins are known for their remarkable stability. For instance, the beta -barrels of FhuA of E. coli (22 beta -strands) or Omp21 from D. acidovorans (8 beta -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 beta -barrel against denaturation is not coupled to localization of those proteins in the outer membrane, since also soluble beta -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 beta -sheet within the MspA monomers is more stable (TM = 112 °C) than the beta -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 beta -barrel domain, which is predominantly held together by hydrogen bonds (44). This fact is also reflected in the pH stability of other beta -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 beta -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 beta -strands. (iv) Proteins located in the inner membrane are always composed of a bundle of hydrophobic alpha -helices, whereas the beta -barrel was exclusively found in outer membrane proteins (13). This was explained by the observation that hydrophobic alpha -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 beta -sheets of the four MspA subunits in a beta -barrel is the easiest model to explain all experimental observations, although the localization and the function of the beta -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).

    ACKNOWLEDGEMENTS

We thank Dr. Wolfgang Hillen for generous support, Dr. W. Curtis Johnson for comments on the CD spectrum, and Danny Beste for reading the manuscript.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant NI 412 and Volkswagen-Stiftung Grant I/77 729.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.

To whom correspondence should be addressed. Tel.: 49-9131-8528989; Fax: 49-9131-8528082; E-mail: mnieder@biologie.uni-erlangen.de.

Published, JBC Papers in Press, December 25, 2002, DOI 10.1074/jbc.M212280200

    ABBREVIATIONS

The abbreviations used are: octyl-POE, n-octylpolyoxyethylene; rMspA, recombinant MspA; Td, dissociation temperature; MES, 2-(N-morpholino)ethanesulfonic acid; ATR, attenuated total reflection; FTIR, Fourier transform infrared.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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