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Department of Biochemistry, Groninger Biomolecular Sciences and Biotechnology Institute, and Materials Science Centreplus (MSCplus), University of Groningen, 9747 AG, Groningen, The Netherlands
Reprint requests to: Bert Poolman, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands; e-mail: B.Poolman{at}rug.nl; fax: +31-50-3634165.
(RECEIVED June 30, 2005; FINAL REVISION September 23, 2005; ACCEPTED September 27, 2005)
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Keywords: MscL; oligomeric structure; covalently linked oligomer; structure/function studies; membrane proteins
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051679005.
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Introduction |
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When MscL activity was first described and the corresponding gene identified in E. coli (Sukharev et al. 1994), it was clear that the channel, with a conductance of 2.5–4 nS, had to be an oligomer of the relatively small (136 amino acids) polypeptide. Moreover, octyl-
-glucopyranoside-solubilized MscL migrated through a gel filtration column as a complex of 60–80 kDa rather than the expected 17 kDa predicted for a monomeric species. On the basis of these experiments, a tetrameric channel was proposed (Hase et al. 1995). However, the exact number of subunits per functional channel has been a matter of discussion ever since.
The response of MscL to sustained membrane tension is different when a small number of channels (an activity burst is followed by inactivation) instead of a large number of channels (sustained or increasing channel activity) is present in a patch. From these observations it has been suggested that the channels assemble in the membrane upon application of mechanical force (Hase et al. 1995), but conclusive experiments have never been presented. Studies in which wild-type MscL and a dimeric version of the polypeptide were subjected to cross-linking resulted in the proposition that the functional MscL unit consisted of six subunits (Blount et al. 1996a, Hase et al. 1997). This hexameric conformation was also supported by a two-dimensional crystallization/ electron microscopy study (Saint et al. 1998).
A major breakthrough came when the structure of MscL from Mycobacterium tuberculosis was determined by X-ray crystallography (Chang et al. 1998). This not only gave insight into the pore structure and potential gating mechanism but also showed that the M. tuberculosis channel consists of five subunits. Subsequently, the quaternary structure of MscL from E. coli was examined by cross-linking, size exclusion chromatography (SEC), and analytical ultracentrifugation, using the wild-type, dimeric, and trimeric tandem constructs. Although the reported analytical ultracentrifugation, cross-linking, and SEC data are not unambiguous, this work is most consistent with the proposal that MscL from E. coli also forms a channel of five subunits (Sukharev et al. 1999). More recently, an electron microscopic study on a homogeneous population of MscL particles confirmed a pentameric structure (Becker et al. 2004).
Although there is no evidence that the oligomeric structure of MscL differs from species to species, such information does exist for other membrane-bound protein complexes, e.g., the FIFO-ATP synthase (in yeast it contains 10 c subunits; in chloroplasts, 14; and in Ilyobacter tartaricus, 11) (Stahlberg et al. 2001). Recently, pore-forming toxins were identified in Staphylococcus aureus that vary in their subunit composition. These pores consist of subunits of LukF and Hlg2 in either a 3:4 or a 4:3 ratio and are thought to play a role in hemolysis of human erythrocytes (Sugawara-Tomita et al. 2002). Both these examples demonstrate that oligomeric structures of proteins (or protein complexes) may differ between species and even within a single species.
Homo-oligomeric structures complicate the analysis of the contribution of individual subunits in the protein function, because it is not possible to engineer single mutations per functional unit. To overcome this problem, Liman et al. (1992) covalently linked oligomers of the mammalian K+ channel. Here, we present the engineering of a covalently linked pentameric MscL channel, which offers unique possibilities for further studies of channel gating by making single or multiple amino acid substitutions or introducing pairs of fluorophores at specific positions in the channel. The covalently linked oligomers also provide in vivo evidence for the pentameric structure of MscL from E. coli.
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Results |
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). The genes were placed under control of the tightly regulated arabinose promoter (PBAD) (Guzman et al. 1995). The gene constructs were designed with unique restriction sites between the individual genes, which allow easy engineering of mutations by exchange of one or more gene fragments. The added base pairs yield insertion of two amino acids between the individual MscL units. The linkers were kept short because previous work had shown that di- and trimeric tandem constructs are active with linkers of only a single amino acid (Blount et al. 1996a, Sukharev et al. 1999). The engineering strategy results in N-terminal extensions for the mono-, di-, and trimeric constructs as indicated in Figure 1. Finally, the last gene of each construct was extended with a sequence specifying a 10-histidine tag.
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. Except for the tetrameric fusion, translation of the constructs started from the ATG codon at the beginning of the multiple cloning site in pBAD, resulting in different N-terminal extensions relative to wild-type MscL for the mono-, di-, and trimeric constructs (Fig. 1A
). As shown in Figure 1B, the insertion of the mscL2 gene into the SacI site resulted in a single base-pair frame shift, relative to the beginning of the multiple cloning site in pBAD at 11 nt downstream of the ATG translation initiation codon. Most likely, the tetrameric construct is translated from the second ATG and starts, like the wild-type, pentameric and hexameric constructs of MscL, with the sequence MSII (Fig. 1B). The final level of expression of the tetrameric construct was lower than that of the other constructs (Fig. 2), probably because the distance between the Shine-Delgarno sequence and translation initiation of mscL2 is too long. It is also possible that an alternative weaker Shine-Delgarno sequence closer to the mscL2 ATG is used (Fig. 1B).
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All tandem constructs encode functional channels
Patch clamp experiments with spheroplasts expressing the different MscL tandem constructs revealed that all covalent oligomers formed active mechanosensitive channels with a unitary conductance of ~3.0 nS (Fig. 3). A typical full electrophysiological recording of a spheroplast expressing wild-type MscS and the monomeric MscL construct is shown in Figure 3A. As the pressure in the pipette was increased, initially only opening (and closing) of MscS was observed. At ~1.7 times the pressure at which two simultaneous MscS openings were observed, the first full opening of MscL was recorded (indicated by an asterisk). In the empty plasmid control, indicated by 0 in Figure 3B, only MscS activity was observed. The number of channels observed per patch was generally between one and five for all the multimeric constructs except for the tetrameric fusion, where in a number of patches no channel activity was observed, which is in accordance with the lower expression level (Fig. 2). Dwell times and substate preference of the oligomeric tandem channels were found not to differ significantly from those of the WT channel (data not shown). This is in contrast to previous findings (Sukharev et al. 1999), where it was reported that dimeric and trimeric tandems with one -amino acid linkers showed a slower open-to-closed transition. It seems that the two amino acid linkers used in our constructs were sufficient to minimize the constraints of covalently linking subunits, resulting in opening and closing characteristics similar to that of the wild-type MscL.
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). Importantly, the pressure ratio of the pentameric tandem construct (1.60±0.09) was similar to that of the wild-type MscL, strongly suggesting that it represents the native oligomeric state.
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Discussion |
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), it can be concluded that the covalently-linked pentameric construct behaves similar to the wild-type MscL channel. The higher pressure ratio required for the opening of the covalent dimeric, trimeric, tetrameric, and hexameric constructs may be explained from the way these polypeptides form functional channels (Fig. 5). If it is assumed that one functional channel comprises of five subunits, one or more nonparticipating subunits must lie at the periphery of the channel in the case of the covalent dimeric, trimeric, tetrameric, and hexameric constructs, as originally suggested by Sukharev and colleagues (Sukharev et al. 1999). We propose that in the dimeric, trimeric, and hexameric constructs, one of the linked subunits is not participating in channel formation and is located at the periphery of the channel (Fig. 5B,C,F). For the tetrameric construct, it would imply that three subunits are not participating in channel formation (Fig. 5D). It is actually surprising that these constructs form functional channels, with relatively little detrimental effects of the nonparticipating subunits. The peripherally-located subunits, however, do hinder the opening of the channel, as can be inferred from the increased pressures needed for initial MscL gating (Fig. 4). This implies that the presence of subunits, which do not participate in the formation of the pore, requires additional energy for the cooperative opening of the channel. The linkers between the subunits may also influence the pressure ratios by restricting the channel flexibility. However, we feel that this is not likely as the pentameric tandem behaves like wild-type MscL.
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, but in a conformation in which all six subunits participate in channel formation. In that case, the sixth subunit would lead to a 1.2-fold increase of the circumference of the channel and possibly a 1.2-fold increase in the pore diameter as compared to the suggested pentameric structure. The conductivity of such a channel could be calculated from the following equation (Hille 1968):
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This equation relates the channel resistance (Rc, which is the reciprocal of the conductance) to the pore radius (r), the pore length (l), and the conductivity of the solution inside the channel (
). Using previously proposed values (Cruickshank et al. 1997) for channel length (l=42Å) and recording solution conductivity (=0.5 
m), the pore radius of a pentameric channel of 3.0 nS conductance would be 18.2 Å. If we increase this radius by a factor of 1.2, the conductance of the hexameric channel would be ~4.0 nS. Such an increase in conductance would have been noticed with our experimental set-up but was not observed. We therefore propose that the hexameric construct also forms a channel with a pentameric structure, that is, with one subunit not participating in the channel. Along the sameline of reasoning, it can be concluded that the dimeric, trimeric, and tetrameric constructs must be composed of the number of polypeptide chains indicated in Figure 5, B and C.
Implications of linker length for the structure of the pentameric construct
In the covalently-linked MscL constructs, the C terminus of MscL 1–4 is conjugated to the N terminus of the next MscL by a linker of only two amino acids. As all constructs were able to form functional channels, this implies that in the open and closed conformations, the N and C termini of the protein can be close together. This is in contrast to the situation in the three-dimensional structure of MscL from M. tuberculosis, where the N and C termini are ~35 Å apart (Chang et al. 1998). In E. coli this distance may be different, as MscL from E. coli is 15 amino acids shorter than MscL from M. tuberculosis. Studies in which the C-terminal helices of MscL from E. coli were subjected to cysteine cross-linking with the channel in the open or closed state (Anishkin et al. 2003) showed that these helices form a rigid helix bundle that is probably directed toward the cytoplasm. It is also known that MscL tolerates relatively large truncations of up to 27 residues from the C terminus (Blount et al. 1996b), and separately expressed N- and C-terminal halves of the protein still form functional channels (Park et al. 2004). This, together with our results, suggests that the position and structure of the C terminus is not very critical for the formation of functional channels.
In summary, we have shown that covalently-linked multimeric constructs of up to six MscL proteins are capable of forming functional pores and that the pentameric construct resembles wild-type MscL in its channel properties. We conclude that the oligomeric state of MscL from E. coli is pentameric. The pentameric gene cassette with unique restriction sites between the individual genes allows easy exchange of wild-type for mutant alleles. The covalent pentameric construct represents an important tool for analyzing the effect on channel function, of single and multiple amino acid substitutions per pore unit, rather than per subunit.
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Materials and methods |
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mscL::Cmres) as a host for the recombinant plasmids (Ou et al. 1998). Cells were grown under aerating conditions at 37°C in Luria Broth, supplemented with 100 µg/mL ampicillin and 30 µg/mL chloramphenicol. To search for optimal expression conditions of mscL, temperatures of 25°C and 30°C and different L-arabinose concentrations were tested. For growth on solid medium, 1.5% (w/v) agar was added to the broth.
Construction of plasmids
The plasmids used in this study are listed in Table 1. The tandem constructs were made by ligating mscL genes one by one into the pBADmyc-HisB vector as shown in Figure 1A. The individual genes were amplified by PCR, using the primers listed in Table 2. During PCR amplification, restriction sites were created upstream and downstream of the mscL gene for directional insertion and positioning of the DNA into the vector. PCR amplifications, using Expand High-Fidelity DNA polymerase (Roche Applied Science) were performed according to the manufacturer’s instructions. The DNA was amplified by PCR using pB10bMscL as template, an annealing temperature of 53°C, and an elongation time of 60 sec.
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and in Table 2, resulting in pBADMscL345, pBADMscL2345, and pBADMscL12345, respectively. To introduce the sixth mscL gene into pBADMscL12345, the fifth gene (mscL1) was replaced by a two-gene fusion (mscL1AB) constructed in pGFPCR. First, mscL1B was inserted into pGFPCR, using XhoI and SacI restriction sites, and then mscL1A was inserted, using NcoI and XhoI restriction sites, resulting in pGFPCRMscL1AB. The oligomeric tandem constructs were used to transform E. coli PB104, and after isolation and purification of the plasmids (Qiagen), the DNA sequence was analyzed to confirm fidelity. Sequencing showed that mscL5 contained a silent mutation. In addition, mscL4 contained a methionine to leucine substitution at position 1 of the MscL coding region.
Expression
E. coli PB104 containing the plasmids with the mscL tandem constructs were grown in LB medium to an OD600 of 0.8, after which expression was triggered by addition of L-arabinose (0.0025% - 0.25% w/v) and induction for different time periods as suggested in Guzman et al. (1995). Inside-out membrane vesicles were prepared by lysing the bacteria (20 mg/mL protein) by a twofold passage through a French pressure cell at 10,000 psi and removal of unlysed cells and cell wall debris by centrifugation at 30,000g. The membrane vesicles were washed once by centrifugation at 150,000g and then resuspended in 50 mM KPi (pH 6.5). Aliquots of 0.5 mL were frozen in liquid nitrogen and stored at –80°C until use.
Spheroplast preparation
Giant spheroplasts were prepared from E. coli PB104, carrying the plasmids with the mscL oligomeric tandem constructs, essentially as described (Blount et al. 1999), except that protein synthesis was triggered using 0.25% L-arabinose for 30 min.
Electrophysiology
Experiments were performed as described previously (Blount et al. 1999; Folgering et al. 2005). Samples of 1–5 µL of spheroplast (0.2 - 0.8 mg/mL total protein) were transferred to a sample chamber containing a ground electrode and 300 µL of patch clamp buffer: 5 mM HEPES (pH 7.2), 200 mM KCl, 90 mM MgCl2, plus 10 mM CaCl2. Channel activity was recorded using an Axopatch 200A amplifier together with a digital converter and Axoscope software (Axon Instruments). Data were acquired at a sampling rate of 33 kHz and filtered at 10 kHz. Offline analysis was performed using Clampfit 8.0 software (Axon Instruments ). Traces shown in Figure 3 were filtered with the low-pass Boxcar filter at smoothing point 7. Pressure ratios for activation of MscL relative to the activation of MscS were determined, as described previously (Blount et al. 1996b, 1999). Briefly, the pressure for MscS gating was defined as the pressure required for the simultaneous opening of two or more channels. The MscL opening threshold was defined as the pressure at which the first full opening was observed.
Miscellaneous
Purified proteins were analyzed on 5%–20% polyacrylamide gradient SDS–PAGE (Laemmli et al. 1970). Protein expression levels in membrane vesicles were analyzed by immunodetection, using antibodies raised against the his-tag (Amersham) and the Western-light chemoluminescence detection kit (Tropix Inc.).
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Acknowledgments |
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References |
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–amino acid sequences of the linkers of the multimeric tandem constructs are indicated. The monomeric, dimeric, and trimeric tandem constructs each start at the ATG of the NcoI restriction site in the plasmid and therefore contain N-terminal extensions as indicated. Each box represents the full sequence of one wild-type MscL from E. coli; the numbers inside the boxes indicate the number of the MscL units (Tables 1
, indicates first opening of MscS; *, indicates first opening of MscL. (B) Typical electrophysiological recordings of spheroplasts from E. coli PB104 expressing the different multimeric constructs. Patches were not saturated, but tension was set to a level where only one or two channels were activated. Time and current scale bars for all traces are indicated at the top. All traces are marked according to the number of MscL units in the construct as in Figure 2
