Correspondence to: Eduardo Perozo, Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, 22906. Fax:(434) 982-1616 E-mail: eperozo@virginia.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O2 or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 2126 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.
Key Words: mechanosensitive channel, solvent accessibility, dipolar coupling, EPR, transmembrane, segments
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MscL, the large conductance mechanosensitive (MS)* channel, provides prokaryotes with a safeguard mechanism in response to hypoosmotic challenges (
Our understanding of the molecular mechanisms of mechanosensation has been significantly advanced with the recent elucidation of the crystal structure of the MS channel from Mycobacterium tuberculosis (Tb-MscL) by
|
In this study, we describe the structural characterization of TM1 and TM2, the two transmembrane domains of the Escherichia coli MscL (Eco-MscL) by site-directed spin labeling and EPR spectroscopy on channels reconstituted in artificial liposomes. Our study demonstrates that the secondary structure and membrane topology of Eco-MscL largely corresponds to the 3-D crystal structure of Tb-MscL. Nevertheless, we also found a few systematic discrepancies between the structures of Eco-MscL and Tb-MscL toward the COOH-terminal end of TM2, suggesting that the cytoplasmic regions of MscL might have a different conformation under physiological conditions. These discrepancies are discussed in conjunction with the differences in the experimental approaches used for the structure determination of the two channel homologues. Preliminary results from this study have been reported in abstract form (
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutagenesis, Expression, and Spin Labeling of MscL
Cysteine mutants were generated for residues 1443 and 72107 in MscL, covering the predicted length of TM1 and TM2, respectively. Mutagenesis was performed by oligonucleotide mismatch site-directed mutagenesis using the Transformer kit (CLONTECH Laboratories, Inc.) and confirmed by dideoxy DNA sequencing. Mutant channels were expressed and purified as follows: the construct MscL-pQE32 containing MscL with the RGS-(4x His) epitope at the NH2 terminus was used to transform E. coli XL-1 blue cells (Stratagene) using standard chemical methods. After protein expression was induced by addition of 1 mM IPTG, membranes were solubilized in PBS containing dodecyl maltoside (DDM) at room temperature, spun-down at 100,000 g for 1 h and purified with a Co2+-based metal-chelate chromatography resin (Talon resin; CLONTECH Laboratories, Inc.). Unless specifically noted, the purified mutant protein was spin labeled overnight with methanethiosulfonate spin label (Toronto Research) at a 10:1 label/channel molar ratio and reconstituted at a 500:1 lipid/channel molar ratio by dilution in PBS (
EPR Spectroscopy and Data Analysis
EPR spectroscopy was performed as described previously ( (
reports on the individual collision frequency of a nitroxide spin label with paramagnetic test compounds, and it is derived from the midpoints of signal saturation at increasing microwave powers according to:
P1/2(X) is the microwave power at which the peak-to-peak amplitude of the MI = 0 EPR resonant line (the central line), I is half-saturated, P is the microwave power in milliwatts, A is a scaling factor, and is a homogeneity parameter. P1/2(X) is measured in the presence of soluble relaxing agents and P1/2(N2) represents the saturation behavior in the absence of paramagnetic collisions. Power saturation curves were obtained for each spin-labeled mutant after equilibration in N2 as control, and air (21% O2), and N2 in the presence of 200 mM Ni-Edda as relaxing agents. All EPR data were obtained at room temperature.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig 1 A shows a schematic representation of an E. coli MscL subunit, where individual rectangles represent the approximate assignments of the -helical elements according to the Tb-MscL crystal structure. Double arrows depict the extent and location of the cysteine mutant set that forms the basis of this study. There is 36% sequence identity between MS channels from E. coli and M. tuberculosis. Since they also differ in the total number of residues (136 and 151, respectively), a baseline correspondence between these two sequences is required to support structural comparisons between the present experimental results and the crystal structure. This numerical equivalence is shown in Fig 1 B, where a ClustalW (
A total of 66 cysteine mutants comprising both transmembrane segments were used in the present study (30 mutants in TM1, and 36 in TM2). Their equivalent position in the Tb-MscL structure is depicted by black spheres on a ribbon diagram of the monomer (Fig 1 C). Each mutant was expressed, purified, labeled, and reconstituted into asolectin liposomes before their analysis by EPR spectroscopy. In the vast majority of cases, expression levels of the cysteine mutants were essentially identical to that of the wild-type channel. At some positions, cysteine mutagenesis produced a moderate (residues 22 and 26 in TM1; and residues 72, 92, and 95 in TM2) to severe (residues 43, 76, and 78) reduction in expression levels. However, in all cases, E. coli growth rates remained unchanged, suggesting that none of the cysteine mutants produced significant gain-of-function effects in MscL. This result confirms recent functional analysis on MscL cysteine mutants (
The structural and membrane topological study of both MscL TM segments was based on an analysis of spectral line shape properties on a per-residue basis. Motional information was obtained from an empiric analysis of the EPR line shape reflecting the degree of motional restriction of the nitroxide spin label, and quantified using the inverse of the width of the central resonance line H0-1 (
O2) is indicative of a residue exposed to membrane lipids; residues exposed to the aqueous environment display high NiEdda (
NiEdda) accessibilities.
Additionally, an estimation of intersubunit proximities was performed from a qualitative analysis of electron spinspin interactions. Through-space spinspin coupling is estimated from the degree of spectral broadening present in samples with two or more paramagnetic centers, compared with that of each individual spectra. It has a distance dependence proportional to R-3 with a practical range between 8 and
25 Å. Unfortunately, in a system with multiple potential labeling sites as MscL, only proximity estimates can be obtained. These types of data form the core of most EPR-based structural analysis, from which features related to a protein secondary and/or tertiary structure can be deduced (for reviews see
TM1 Residue Environmental Parameters
Fig 2 shows the complete spectral dataset for TM1. An overview of the different types of observed line-shapes reveals a six-residue segment (positions 2127) in which the spectra appear considerably broadened. Evaluation of the mobility parameter Ho-1 from the EPR spectra obtained for the TM1 segment confirms this impression (Fig 3, top). Additionally, it reveals a strong periodicity in the mobility of the spin label from neighboring amino acid residues, a behavior consistent with the observed
-helical structure of the first transmembrane domain in the crystal structure (L19A38 in Tb-MscL). This periodicity includes even the highly immobilized stretch of residues 2127 (although with a reduced dynamic range). In spite of the closed packing arrangement for the TM1 segment seen in the Tb-MscL crystal structure, starting at position 29, there is a gradual increase in the mobility parameter through I40. We have interpreted this observation to suggest that the spin labels at these locations are able to resolve the subtle changes in motional dynamics derived from the gradual increase in the diameter of the outer vestibule of the channel. A similar mobility gradient is observed in TM2 and correlates well with the idea that the most immobilized regions of the channel are toward the cytoplasmic ends (where the gate is proposed to be).
|
|
The periodic structure of the TM1 segment is also reflected in the O2 accessibility profile, where the data shows very low O2 collision frequencies for every third or fourth residue along the entire TM1 segment (Fig 3, middle). Interestingly, significant rates of O2 collision are observed even within the highly immobilized region comprised by residues 2127. As seen in other membrane proteins (for reviews see
The NiEdda accessibility profile correlates well with the mobility data, revealing a region of eight residues (positions 1926), for which accessibility to NiEdda is severely restricted (Fig 3, bottom). This result is in very good agreement with the Tb-MscL crystal structure. Residues in this region of the channel form a hydrophobic constriction zone forming the narrowest point along the permeation path with a diameter of 2 Å. This area is likely to form a significant barrier to the passage of ions and other solutes, thus, contributing to form the "gate" that physically closes MscL channels.
A remarkable observation, also derived from the NiEdda accessibility profile, is the presence of residues along TM1 with significant (even high) NiEdda collision frequencies (positions 28, 31, 35, and 36). These residues are located well within the membrane-embedded portions of TM1, and are a clear manifestation of the large water-filled extracellular vestibule observed in the Tb-MscL crystal structure. The fact that the O2 (a probe of hydrophobicity) and NiEdda (a probe of hydrophilicity) profiles are essentially 180° out of phase confirms that the TM1 segment simultaneously surrounds an aqueous pore while exposed to the membrane phospholipids. This is clear only for the COOH-terminal half of the helix, as increasing proteinprotein contacts among TM1 segments and between TM1 and TM2 segments significantly restrict solvent accessibility in the remainder of the helix.
The correlation between the EPR-derived environmental parameters and their location within the crystal structure is shown in Fig 4, where the unprocessed data for H0-1,
O2, and
NiEdda are mapped onto their equivalent positions in Tb-MscL. In each case, the property mapped is shown in the context of a solvent-accessible surface representation (1.4-Å probe size) of the full-length channel, and as solvent-accessible surface of TM1 only (in two orientations, rotated 180°).
|
TM2 Residue Environmental Parameters
Fig 5 shows a family of EPR spectra from residues V72 to E107, encompassing the length of the TM2 segment and a portion of the intracellular loop. From these spectral line-shapes, it appears that this segment, as a whole, is slightly less motionally restricted than TM1. This is to be expected from a region of the channel that is predicted to contact the more motionally dynamic environment of the membrane lipids. As is typically the case for protein regions that contact only two different environments, the Ho-1 profile (Fig 6, top) shows a remarkably periodic behavior. When analyzed by Fourier methods, the power spectrum for this mobility profile (residues 7296) reveals a single peak centered at 102° and with a periodicity index of 3.67 (data not shown), in broad agreement with the structure of TM2 derived from the X-ray data (
O2 (Fig 6, middle), confirming not only that TM2 is at the periphery of the channel, but also that the majority of its residues are directly in contact with the membrane lipid.
|
|
Equivalent to what is seen at the COOH-terminal half of TM1, high NiEdda collision frequencies are observed at the NH2-terminal end of TM2, a region expected to be fully embedded in a hydrophobic environment (Fig 6, bottom panel). In both cases, this pattern of accessibility to the aqueous medium occurs 180° out of phase with the O2 profile, and originates from the presence of the large water-filled vestibule in the extracellular region of the channel. NiEdda accessibility decreases dramatically toward the COOH-terminal end of TM2, reflecting the local environment of residues that contact either lipid or neighboring TM1 segments. A sharp increase in NiEdda accessibility for residue 100 signals the likely point where this TM segment exits the membrane interface. Yet, position 96 corresponds to the last truly helical residue as described in the Tb-MscL structure. This segment is immediately followed by a region of progressively larger motional freedom, very high NiEdda accessibility and relatively flat O2 accessibility, all diagnostic properties of aqueous loop-like structures. These points are well illustrated in Fig 7, where EPR-derived residue environmental data are mapped onto the Tb-MscL crystal structure (parameters of the probe size and color mapping are identical to those of Fig 4). In the case of the
H0-1 and
O2 maps, the dotted squares points to residues in TM2 where experimental values were found to be much lower than expected (positions 93 and 94).
|
As shown by , the natural log of the ratio between the
P1/2 obtained in the presence of O2 and in the presence of NiEdda, is directly proportional to the immersion distance from the membranewater interface. Thus, with proper calibrations, this value can be used to estimate immersion depths for a series of solvent-exposed residues in a given transmembrane segment. Calculated only for TM2 residues with high collision rates (Fig 8), these depth estimates strongly suggest that MscL can be positioned within the context of a membrane bilayer with residues 8687 at or very close to the center of the bilayer.
|
Intersubunit SpinSpin Interactions
In an oligomer with radial symmetry and a single spin label per subunit, only those residues in close proximity to the symmetry axis are expected to show strong spinspin coupling. Because of the fivefold symmetry in MscL, the spectral broadening from fully labeled channels originates not only from the spinspin interactions between a given subunit and its immediate neighbors, but also from diagonally related subunits at two different positions. Therefore, spectral broadening derived from spin coupling cannot be easily used in the determination of actual intersubunit distances. In this situation, rather than estimate individual distances, one can establish the unique pattern of proximities present in a given multisubunit system. A gross indication of intersubunit proximities can be obtained from the parameter (
Maximally labeled and underlabeled spectra from selected residues along TM1 (Fig 9 A) illustrate the point. When labeled at a 10:1 molar ratio of MscL monomer to spin label, the vast majority of the labeled channels are predicted to have a single nitroxide group and, hence, represents the unbroadened spectra (Fig 9 A, red traces). Reversing the labeling ratio (1:10 molar ratio, monomer to spin label) maximizes the likelihood that all available cysteines will be modified with a spin label, thus, assuring broadened spectra (Fig 9 A, black traces). Application of this type of analysis generates an intersubunit interaction profile (Fig 9) pointing to residues likely to be located near the symmetry axis of MscL (its permeation path). Results from the TM1 segment (Fig 9, top) reveal a prominent cluster of interacting residues at positions 2026, in remarkable agreement with the mobility and NiEdda accessibility data (Fig 3, top and bottom), suggesting a hydrophobic constriction among these residues. Significant levels of spectral broadening were also observed, with an impressive -periodicity, at both sides of the major cluster. Surprisingly, we were able to detect small, but significant, levels of spinspin interactions among residues in the COOH-terminal half of TM2, suggesting that this side of the TM2 helix is likely to face the symmetry axis (Fig 9, bottom). The spatial distribution of these interacting residues within MscL is illustrated in Fig 9 C, where
values obtained for residues in both TM segments are mapped into the Tb-MscL structure, either in ribbon representation or in a cross-section of an accessible surface representation. It is worth noting that the strongest
values from the 20 to 26 cluster forms an unmistakable ring around the narrowest section of the MscL permeation path, strongly suggesting that this section of the channel physically contributes to restrict solute permeation in the closed state.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The main goal of the present study was to provide an initial assessment of the structural dynamic properties of MscL channels in the closed state. This was done using spin labels to report on the local residue environmental properties along the two transmembrane segments of Eco-MscL reconstituted in lipid vesicles. This information was then compared with the current crystallographic model from Tb-MscL to establish the relevance of this model to the configuration of the closed channel under physiological conditions.
An examination of Fig 4 and Fig 7 suggests that our solvent accessibility determinations, when mapped onto the Tb-MscL crystal structure, conform quite well to expected patterns of surface distribution. The only apparent exception being the unusually low accessibility values for residues located at the COOH-terminal end of TM2. A quantitative comparison between these two structural views of MscL can be obtained by contrasting the static solvent-exposed areas of individual residues (calculated from the crystal structure), with their EPR experimentally determined values (Fig 10). Solvent-exposed areas were obtained using an algorithm based on the Gauss-Bonnet theorem (GETAREA;
|
A similar analysis performed on TM2 produced excellent agreement between experimental and calculated accessibilities for a large section of the helix (residues 72100), but also revealed a major discrepancy for residues 9195 (Fig 10 B; see also Fig 4). These results can be explained as either a phase change from the preceding -periodicity in residues 7290, the consequence of a tightly bound molecule within the membrane-embedded regions of the channel, or as the result of proteinprotein interaction with other elements of the channel. Furthermore, a review of the spectral line-shapes of residues F93, M94, and A95 also suggest an incompatibility between their dynamic behavior, predicted from the crystal structure, and the present EPR data (Fig 10 C). Perhaps the most extreme example is residue M94, where the crystal structure predicts a fully extended side chain, away from the symmetry axis and toward the membrane lipids, yet the EPR spectrum is almost as immobilized as in the rigid limit.
What might be the origin of this discrepancy? The presence of additional electron density surrounding Tb-MscL crystals (Chang, G., R.H. Spencer, and D.C. Rees, personal communication) has been suggested to correspond to immobilized lipids. If the binding interaction of these molecules is extensive enough, and its dynamics slow in the EPR time scale, these lipid molecules could partially explain the discrepancies in nitroxide mobilities for residues in the last turn of the TM2 helix. However, it would be more difficult to explain the observed disagreement in the accessibility values. The obvious alternative explanation requires the existence of proteinprotein interactions not observed in the present MscL crystal structure. Clearly, without further EPR analysis of additional regions in MscL it is difficult to pinpoint the actual cause of these differences. However, an obvious possibility is that the cytoplasmic regions of the channel might physically interact with TM2 near the membrane interface. One candidate could be the NH2-terminal end, a segment of 12 residues that is not resolved from the present crystallographic data. Alternatively, the COOH terminus might not assemble into the cytoplasmic five-helix bundle observed in the crystal structure and, instead, it could fold toward the membrane interface, establishing specific tertiary contacts with TM2. It is important to note that the Tb-MscL crystallization experiments were performed at a very low pH (between 3.6 and 3.8;
Not counting the contributions of the NH2 terminus, the cytoplasmic-exposed regions of MscL are highly charged (9 basic and 7 acidic residues in Eco-MscL), and some of these residues form part of a highly conserved charged cluster of residues present not only in all the MscL homologues identified to date, but also in all members of the recently identified family of prokaryotic MS channels (
It is important to define which regions of MscL physically contribute to form the gate that so effectively blocks solute flow at rest, but is able to undergo such massive conformational change that leads to an open conduction pore of 3040 Å in diameter (35-Å thickness of the membrane bilayer.
A recent model for the gating mechanism of MscL introduced the notion that the NH2 terminus might form a second external gate in addition to the hydrophobic gate formed by the TM1 segments ( calculated for the residues G14 and N15 (Fig 9 B), which according to the model form a part of the S1-M1 linker, indicates some degree of spinspin coupling for these residues, as expected for regions of the channel adjacent to the permeation path. The mobility and structural role of the NH2 termini in the MscL pentamer, however, requires additional analysis for which the present approach appears ideally suited.
Having established a spectroscopic "native dataset" for MscL in the closed configuration, we are in position to undertake an analysis of its gating mechanism from a structural standpoint. Some of the key issues that remain to be established include not only the types and directions of the molecular rearrangements that underlie channel opening, but also the fundamental physical mechanisms that allow the transduction of bilayer deformations into protein movement. It is perhaps the answer to this last question what could make possible a spectroscopic analysis of MscL gating, since a key challenge in such a project is in fact the ability to stabilize the open state under zero transbilayer tensions. It will be critical to understand the extent to which the internal bilayer forces (hydrophobic mismatch, membrane intrinsic curvature) contribute to trigger MscL gating.
In conclusion, we have characterized the structural dynamic properties of the transmembrane elements of MscL using site-directed spin labeling and EPR spectroscopy. Our results establish the overall structural validity of the recent crystal structure as a model for the membrane-embedded regions of MscL in their natural environment. Nonetheless, substantial inconsistencies in residue environmental parameters toward the COOH-terminal end of the TM2 helix seem to suggest that this agreement might not hold toward the soluble, cytoplasmic-facing regions of the channel under physiological conditions. Consequently, our findings emphasize the advantage of alternative (even if lower resolution) methods in the determination of higher order structures of membrane proteins under conditions that closely reflect the protein native membrane environments. Furthermore, this study sets the stage for a structural analysis of the gating mechanism in this class of MS channels.
![]() |
Footnotes |
---|
* Abbreviations used in this paper: EPR, electron paramagnetic resonance; MS, mechanosensitive.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work was made possible due to the financial support from the following sources: the Australian Research Council (grant A00000819 and IP97054 to B. Martinac), the Australian Academy of Science (Scientific Visit Award to B. Martinac), the National Institutes of Health (grants GM636170 to E. Perozo), and the McKnight endowment fund for neuroscience (McKnight Scholar Award to E. Perozo).
Submitted: 1 May 2001
Revised: 6 July 2001
Accepted: 9 July 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altenbach, C., Greenhalgh, D.A., Khorana, H.G., and Hubbell, W.L. 1994. A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc. Natl. Acad. Sci. USA. 91:1667-1671[Abstract].
Batiza, A.F., Rayment, I., and Kung, C. 1999. Channel gate: tension, leak and disclosure. Struct. Fold. Design 7:R99-R103.
Blount, P., Sukharev, S.I., Moe, P.C., Schroeder, M.J., Guy, H.R., and Kung, C. 1996. Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli. EMBO J. 15:4798-4805[Abstract].
Chang, G., Spencer, R.H., Lee, A.T., Barclay, M.T., and Rees, D.C. 1998. Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science. 282:2220-2226
Columbus, L., Kálai, T., Jekö, J., Hideg, K., and Hubbell, W.L. 2001. Molecular motion of spin labeled side chains in -helices: analysis by variation of side chain structure. Biochemistry. 40:3828-3846[Medline].
Cortes, D., Cuello, L., and Perozo, E. 2001. Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating. J. Gen. Physiol. 117:165-180
Cruickshank, C.C., Minchin, R.F., Le Dain, A.C., and Martinac, B. 1997. Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli. Biophys. J. 73:1925-1931[Abstract].
Cuello, L.G., Romero, J.G., Cortes, D.M., and Perozo, E. 1998. pH-dependent gating in the Streptomyces lividans K+ channel. Biochemistry. 37:3229-3236[Medline].
Elmore, D.E., Phillipson, K.D., and Dougherty, D.A. 2001. Molecular dynamics symulations of the M. tuberculosis mechanosensitive channel of large conductance. Biophys. J. 80:110a. (Abstr.).
Farahbakhsh, Z.T., Altenbach, C., and Hubbell, W.L. 1992. Spin labeled cysteines as sensors for protein-lipid interaction and conformation in rhodopsin. Photochem. Photobiol. 56:1019-1033[Medline].
Fraczkiewicz, R., and Braun, W. 1998. Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J. Comp. Chem. 19:319-333.
Hamill, O.P., and McBride, D.W. 1997. Induced membrane hypo/hyper-mechanosensitivity: a limitation of patch-clamp recording. Annu. Rev. Physiol. 59:621-631[Medline].
Hamill, O.P., and Martinac, B. 2001. Molecular basis of mechanotransduction in living cells. Physiol. Rev. 81:685-740
Häse, C.C., Le Dain, A.C., and Martinac, B. 1995. Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli. J. Biol. Chem. 270:18329-18334
Hubbell, W.L., Cafiso, D.S., and Altenbach, C. 2000. Identifying conformational changes with site-directed spin labeling. Nat. Struct. Biol. 7:735-739[Medline].
Kabsch, W., and Sander, C. 1983. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 22:2577-2637[Medline].
Kleywegt, G.J., and Jones, T.A. 1994. Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallographica D. 50:178-185.
Kloda, A., and Martinac, B. 2001. Molecular identification of a mechanosensitive channel in archaea. Biophys. J. 80:229-240
Levina, N., Totemeyer, S., Stokes, N.R., Louis, P., Jones, M.A., and Booth, I.R. 1999. Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 18:1730-1737
Martinac, B. 2001. Mechanosensitive Channels in Prokaryotes. Cell. Physiol. Biochem. 11:61-76[Medline].
Martinac, B., Buechner, M., Delcour, A.H., Adler, J., and Kung, C. 1987. Pressure-sensitive ion channel in Escherichia coli. Proc. Natl. Acad. Sci. USA. 84:2297-2301[Abstract].
Martinac, B., Adler, J., and Kung, C. 1990. Mechanosensitive ion channels of E. coli activated by amphipaths. Nature. 348:261-263[Medline].
Martinac, B., Kloda, A., and Perozo, E. 2000. Structural dynamics of MscL first transmembrane segment. a site directed spin-labeling study. Biophys. J. 78:137a. (Abstr.).
Mchaourab, H., and Perozo, E. 2000. Determination of protein folds and conformational dynamics using spin-labeling EPR Spectroscopy. In Eaton G.E.S., Berliner L., eds. Biological Magnetic Resonance. Vol. 19. New York, Kluwer-Plenum, 155-218.
Mchaourab, H.S., Lietzow, M.A., Hideg, K., and Hubbell, W.L. 1996. Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry. 35:7692-7704[Medline].
Moe, P.C., Blount, P., and Kung, C. 1998. Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation. Mol. Microbiol. 28:583-592[Medline].
Nicholls, A., Sharp, K.A., and Honig, B. 1991. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 11:281-296[Medline].
Oakley, A.J., Martinac, B., and Wilce, M.C. 1999. Structure and function of the bacterial mechanosensitive channel of large conductance. Prot. Sci. 8:1915-1921[Abstract].
Perozo, E., Cortes, D.M., and Cuello, L.G. 1998. Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy. Nat. Struct. Biol. 5:459-469[Medline].
Perozo, E., Cortes, D.M., Kloda, A., and Martinac, B. 2001. Structural dynamics of MscL outer transmembrane segment. A site directed spin-labeling study. Biophys. J. 80:345a. (Abstr.).
Rees, D.C., Chang, G., and Spencer, R.H. 2000. Crystallographic analyses of ion channels: lessons and challenges. J. Biol. Chem. 275:713-716
Spencer, R.H., Chang, G., and Rees, D.C. 1999. Feeling the pressure: structural insights into a gated mechanosensitive channel. Curr. Opin. Struct. Biol. 9:448-454[Medline].
Subczynski, W.K., and Hyde, J.S. 1981. The diffusion-concentration product of oxygen in lipid bilayers using the spin-label T1 method. Biochim. Biophys. Acta. 643:283-291[Medline].
Sukharev, S.I., Martinac, B., Arshavsky, V.Y., and Kung, C. 1993. Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. Biophys. J. 65:177-183[Abstract].
Sukharev, S.I., Blount, P., Martinac, B., Blattner, F.R., and Kung, C. 1994. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature. 368:265-268[Medline].
Sukharev, S.I., Blount, P., Martinac, B., and Kung, C. 1997. Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities. Annu. Rev. Physiol. 59:633-657[Medline].
Sukharev, S.I., Sigurdson, W.J., Kung, C., and Sachs, F. 1999. Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL. J. Gen. Physiol. 113:525-540
Sukharev, S.I., Betanzos, M., Chiang, C.-S., and Guy, H.R. 2001. The gating mechanism of the large mechanosensitive channel MscL. Nature. 409:720-724[Medline].
Thompson, J., Higgins, D.G., and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680[Abstract].
Yoshimura, K., Batiza, A., and Kung, C. 2001. Chemically charging the pore constriction opens the mechanosensitive channel MscL. Biophys. J. 80:2198-2206
Yoshimura, K., Batiza, A., Schroeder, M., Blount, P., and Kung, C. 1999. Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity. Biophys. J. 77:1960-1972