Properties of the Mechanosensitive Channel MscS Pore Revealed by
Tryptophan Scanning Mutagenesis
Tim Rasmussen,
†
Akiko Rasmussen,
†
Shivani Singh,
†
,
§
Heloisa Galbiati,
†
Michelle D. Edwards,
†
Samantha Miller,
†
and Ian R. Booth
*
,
†
,
‡
†
School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom
‡
Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena,
California 91125, United States
*
S
Supporting Information
ABSTRACT:
Bacterial mechanosensitive channels gate when
the transmembrane turgor rises to levels that compromise the
structural integrity of the cell wall. Gating creates a transient large
diameter pore that allows hydrated solutes to pass from the
cytoplasm at rates close to those of di
ff
usion. In the closed
conformation, the channel limits transmembrane solute move-
ment, even that of protons. In the MscS crystal structure (Protein
Data Bank entry 2oau), a narrow, hydrophobic opening is visible
in the crystal structure, and it has been proposed that a vapor
lock created by the hydrophobic seals, L105 and L109, is the
barrier to water and ions. Tryptophan scanning mutagenesis has
proven to be a highly valuable tool for the analysis of channel
structure. Here Trp residues were introduced along the pore-
forming TM3a helix and in selected other parts of the protein.
Mutants were investigated for their expression, stability, and activity and as
fl
uorescent probes of the physical properties along the
length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as
heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low
accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state
fl
uorescence anisotropy data are consistent
with signi
fi
cant homo-Fo
̈
rster resonance energy transfer between tryptophan residues from di
ff
erent subunits within the narrow
pore. The data provide new insights into MscS structure and gating.
M
echanosensitive channels gate in response to increased
tension in the lipid bilayer that arises from lowering of
the external osmolarity (hypoosmotic shock).
1
−
3
A major
structural transition takes place to create a large, transient
transmembrane pore that is fully hydrated,
4
which allows the
transit of small, osmotically active solutes. The pores are usually
relatively nonspeci
fi
c for the solutes, and only molecular
volume is a good predictor of which solutes might pass through
the channel. The channels have been demonstrated to play a
major role in protecting the mechanical integrity of the bacterial
cell during hypoosmotic shock
5
and in stabilizing the structure
of chloroplasts and yeast endoplasmic reticulum.
6
−
8
There are
two major types of channels, MscL (the channel of large
conductance) and MscS (the channel of small conduc-
tance),
5,9,10
where the latter is a large family of proteins with
many structural variations.
11
Through crystallography, molec-
ular dynamics, biophysical approaches, and molecular genetics,
we have come to understand much about the structure of the
pore of MscS and the gating process.
4,12
−
20
Multiple structures of MscS from di
ff
erent organisms in both
nonconducting (closed)
12,21
and conducting (open) con
fi
g-
urations
19,21
(a further structure from
Thermoanaerobacter
tengcongensis
may also be in the closed state
22
) have been
determined. A more condensed state has been proposed on the
basis of molecular dynamics.
17
Central to understanding the
mechanism is the nature of the closed pore. Thus, the crystal
structure shows MscS to be a homoheptamer (and indeed all
MscS variants studied to date exhibit masses consistent with a
heptamer).
23,24
Each MscS subunit has two transmembrane
helices, TM1 and TM2, that form a
“
sensor
”
paddle attached to
the pore-forming helix, TM3a. All seven subunits contribute a
helix to the pore. A pronounced kink after residue Q112
(
Escherichia coli
MscS numbering) leads to an amphipathic
helix, TM3b, that has been predicted to lie at the membrane
interface with its hydrophobic surface interacting with lipid
chains and the hydrophilic face intercalating with the
β
domain.
12
The pore-forming (TM3a) and pore-sensing
domains (TM1 and TM2) are inserted through the lipid
bilayer, such that the amino-terminal residues are integrated
into the headgroup region of the outer lea
fl
et of the membrane,
Received:
March 17, 2015
Revised:
June 18, 2015
Published:
July 1, 2015
Article
pubs.acs.org/biochemistry
© 2015 American Chemical Society
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are periplasmic, or form a cap on the pore helices.
25,26
This
region is not resolved in any of the crystal structures.
12,19,22
The
open pore thus forms a conduit connecting the periplasmic
space with the cytoplasm, the latter being achieved through
portals formed in the large cytoplasmic vestibule.
12
In the original crystal structure of the nonconducting MscS, a
moderately large
“
hole
”
was evident that was, at least partially,
sealed by two rings of leucine residues (L105 and L109,
E. coli
MscS numbering).
12
The appearance of the protein suggested
the possibility that an open form had been captured. However,
measurements of the pore by a variety of methods showed that
its diameter was inconsistent with the known conductance of
the channel.
27
In parallel, it was proposed, from MD
simulations, that the pore was su
ffi
ciently hydrophobic to
create a vapor lock that would prevent a column of water from
forming in the pore and this would block ion conduction.
28,29
Thus, the closed state is nonconducting by virtue of the
hydrophobicity of the residues facing into the pore and the
tight packing of the TM3a helices. When the open structure
was determined,
19
it was clear that the expanded state of the
pore was achieved by straightening the TM3a helices and their
displacement from the pore axis to create a water-
fi
lled pore.
The diameter of this pore was shown to be consistent with the
known conductance.
19
Tryptophan substitution mutagenesis has proven to be a
valuable tool for the analysis of ion channel structure
30
−
34
because the unique
fl
uorescence properties of this amino acid
allow inferences to be drawn about the environment in which
the side chain is located. Tryptophan has the most stringent
space requirements of all natural amino acids not only because
of its large molecular volume (163 Å
3
) but also because of the
rigidity of the aromatic side chain. Accommodation of the Trp
residue in mutant channels may require an altered con-
formation that modi
fi
es the stability and activity of the channel.
However, the susceptibility of Trp
fl
uorescence to lipid-borne
and soluble quenchers provides valuable insights into the
organization of the protein. For MscS, we have previously
shown that Trp can be substituted into the pore for Leu at
positions 105 and 109 and the resulting channels assembled in
the membrane and retained function in physiological assays,
albeit at a lowered level relative to that of the wild type.
15,20
However, when puri
fi
ed, only the L105W variant was stable.
This suggested that Trp substitution mutagenesis would
provide a useful tool for investigating the pore of MscS, in
particular for identifying regions that are sensitive to
perturbation. We have previously reported the properties of a
Trp-free MscS protein, MscSYFF.
20
The mutant channel
possesses protective ability in downshock assays similar to
that of the wild type, but in patch clamp assays, it required a
tension
∼
50% higher than that of the wild type to undergo the
transition to the open state.
20
We used this mutant protein for
construction of Trp substitutions at each position in pore-lining
helix TM3a and at selected positions in TM1 and TM2. In the
closed state, TM3a helices pack tightly with a
“
knobs and
grooves
”
arrangement involving residues 98
−
101, 102
−
104,
and 106
−
108.
12,13
The other residues either face into the pore
or interact with TM1, TM2, or lipids. The data show that
although the structure of MscS may be perturbed by Trp
insertion, most of the mutants form stable heptamers that can
be puri
fi
ed and reconstituted into lipid bilayers. Modi
fi
ed
activity was detected for the mutant channels, both by
electrophysiology and by their protective ability during
hypoosmotic shock; only a few mutant channels exhibited
properties close to those of the wild-type channel in one or
more assays. At each position examined, the Trp
fl
uorescence
exhibits unique properties with respect to emission maximum
and accessibility to water-borne quenching agents and the data
are consistent with a very hydrophobic pore.
■
EXPERIMENTAL PROCEDURES
Materials and Bacterial Strains.
Isopropyl
β
-
D
-thiogalac-
toside (IPTG) was obtained from Melford Laboratories
(Ipswich, U.K.),
n
-dodecyl
β
-
D
-maltopyranoside (DDM) from
A
ff
ymetrix Anatrace (High Wycombe, U.K.), and 1,2-dioleoyl-
sn
-glycero-3-phosphocholine (DOPC) from Avanti (Alabaster,
AL), and all other chemicals were from Sigma-Aldrich
(Gillingham, U.K.).
E. coli
strains MJF429 (Frag1
Δ
mscS,
Δ
mscK::Kan
) and MJF612 (Frag1
Δ
mscL::cm,
Δ
mscS,
Δ
mscK::kan,
Δ
ybdG::apr
) were constructed and characterized
previously.
5,24
Molecular Biology and Physiological Assays.
The
pTrcMscSYFF construct,
20
with mutations W16Y, W240F,
and W251F, was used as a template to introduce tryptophan
mutations by applying the Stratagene QuikChange protocol.
Mutations were con
fi
rmed by sequencing on both DNA
strands. Survival after osmotic downshock was tested as
described previously
14
using transformants of MJF612 grown
in Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L NaCl,
and 5 g/L yeast extract) containing 0.5 M NaCl at 37
°
C. At an
OD
650
of 0.2, expression of the mutated MscS was induced by
addition of 0.3 mM IPTG until the OD
650
reached 0.3. In
parallel, noninduced samples were prepared. The cells were
then diluted in LB medium (shock) or diluted in LB medium
containing 0.5 M NaCl (control). After incubation for 10 min,
serial dilutions were made and spread on LB agar plates with or
without 0.5 M NaCl. The plates were incubated overnight at 37
°
C, and colonies were counted. It should be noted that the
relationship among channel abundance, channel activity, and
survival is a complex one for both MscS
13,14,35
and MscL.
36
A
more complete discussion of the factors determining the
outcome of this assay is provided elsewhere.
35,37
Electro-
physiological experiments were performed as described
previously.
5,13
Giant protoplasts were generated from MJF429
cells transformed with the relevant constructs after induction
with 1 mM IPTG for 15
−
45 min. Excised, inside-out patches of
the protoplasts were analyzed by patch clamp at a membrane
potential of
−
20 mV using the same bu
ff
er on both sides of the
membrane [5 mM HEPES (pH 7.0), 200 mM KCl, 90 mM
MgCl
2
, and 10 mM CaCl
2
]. Recordings were performed with
an AxoPatch 200B ampli
fi
er and pClamp software (Axon) at a
sampling rate of 50 kHz and
fi
ltration of 5 kHz. The pressure
ratios relative to the pressure threshold of MscL (
P
L
:
P
S
) are
given as a relative indicator of the pressure required to open the
mutant forms of MscS as described previously.
38
This analysis
could be applied accurately only to channels that exhibited
frequent stable openings such that the pressure at which two
channels are open simultaneously could be determined.
Puri
fi
cation of MscS Tryptophan Mutants.
Puri
fi
cation
of the MscS tryptophan mutants followed the protocol
established previously.
15,20
Membranes were solubilized by
incubation for 1 h at 4
°
C in 1.5% DDM containing 50 mM
sodium phosphate (pH 7.5), 300 mM NaCl, 10% glycerol, 50
mM imidazole, and 0.2 mM phenylmethanesulfonyl
fl
uoride
(PMSF, Sigma). Aggregates were removed by centrifugation at
3000
g
for 10 min and
fi
ltration using a 0.2
μ
m syringe
fi
lter.
MscS was then bound through its C-terminal His
6
tag to a
Biochemistry
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prepacked 0.5 mL nickel-nitrilotriacetic (Ni-NTA) agarose
column (Sigma) and washed with 20 mL of bu
ff
er A [50 mM
sodium phosphate (pH 7.5) containing 0.05% DDM, 300 mM
NaCl, 10% glycerol, and 50 mM imidazole]. After storage
overnight at 4
°
C, MscS was eluted with elution bu
ff
er B (bu
ff
er
A containing 300 mM imidazole). Peak fractions were
separated on a HiPrep Superdex 200 16/600 size exclusion
column (GE Healthcare) at a rate of 1 mL min
−
1
using a bu
ff
er
containing 0.03% DDM, 50 mM sodium phosphate (pH 7.5),
and 150 mM NaCl. The ratio of heptamers to monomers was
used as a guide to the stability of the mutated channel complex.
For
fl
uorescence measurements in bilayers, MscS was
reconstituted by dilution into DOPC following a protocol
described in detail in ref 39. In short, MscS solubilized in DDM
was mixed with DOPC solubilized in sodium cholate (Sigma)
at a molar ratio of 1:100 and incubated for 15 min at room
temperature. Then the mixture was diluted 30-fold, far below
the critical micelle concentration for the detergents, into the
measuring bu
ff
er containing 40 mM HEPES (pH 7.2), 100 mM
KCl, and 1 mM EGTA, and emission spectra were recorded
after equilibration for 5 min.
Blue Native Polyacrylamide Gel Electrophoresis (BN-
PAGE).
MscS constructs were transformed into MJF612 and
grown to an OD
650
of 0.4 in 120 mL of LB medium. MscS was
induced by addition of 0.3 mM IPTG and incubation continued
for 30 min at 37
°
C. Expression of mutant MscS channels in
this study did not cause impaired growth upon induction of the
transformed
E. coli
strain. Cells were harvested by centrifuga-
tion and kept at
−
80
°
C until further use. The cell pellet was
suspended in 5 mL of PBS bu
ff
er [137 mM NaCl, 2.7 mM KCl,
10 mM Na
2
HPO
4
,and2mMKH
2
PO
4
(pH 7.4)]
supplemented with 0.2 mM phenylmethanesulfonyl
fl
uoride
(PMSF) and lysed by a single passage through a French press at
18000 psi. After centrifugation at 3000
g
for 20 min, 3.2 mL of
the supernatant was centrifuged at 100000
g
for 1 h at 4
°
C. The
membrane pellet was suspended in 125
μ
L of PBS and frozen
in small aliquots at
−
80
°
C until further use. The total protein
concentration was determined by the Lowry assay.
40
Mem-
brane suspensions corresponding to 30
μ
g of protein were
solubilized in PBS bu
ff
er (pH 7.4) containing 1% DDM, 2.8 M
urea, and 1 mM EDTA for 30 min at room temperature.
Samples were centrifuged at 14000
g
for 10 min at 4
°
C. After
addition of native sample bu
ff
er and Coomassie G-250 [as per
the manufacturer
’
s instructions (Invitrogen)] to the super-
natant, samples were loaded onto Novex 4 to 16% Bis-Tris
gradient native gels (Invitrogen) and run at 150 V for 2 h.
Details of the gel PAGE and the Western blotting followed the
protocol of the supplier (Invitrogen).
Fluorescence Spectroscopy.
Fluorescence spectroscopy
was performed as described in detail previously
15,20
using an
FLS920
fl
uorescence spectrometer from Edinburgh Instru-
ments (Livingston, U.K.). For steady-state emission spectra,
tryptophan was excited at 295 nm with a slit width of 2 nm and
the emission was recorded between 300 and 420 nm with a slit
width of 2 nm. Reconstituted samples were measured with slit
widths of 3 and 7 nm. A 0.2 mL micro cuvette with 10 mm
excitation and 4 mm emission path lengths was used (Hellma).
The excitation polarizer was set to 90
°
and the emission
polarizer to 0
°
.
41
The spectrum was
fi
tted to a skewed Gaussian
after correction with a bu
ff
er spectrum:
λλω
=−+−
+
λ
I
Ibb
a
exp(( ln 2){ln[1 2 (
)/ ]/ } )
max
max
2
(1)
where
I
is the wavelength-dependent
fl
uorescence intensity,
I
max
the maximal intensity,
b
a skewing factor,
λ
the emission
wavelength,
λ
max
the wavelength of the peak, and
ω
λ
the width
of the peak at half-maximal intensity. Quenching by acrylamide
in the concentration range of 0
−
0.2 M was quanti
fi
ed using the
Stern
−
Volmer equation:
=+
I
IK
/1 [Q
]
0SV
(2)
where
I
0
and
I
are the
fl
uorescence intensities in the absence
and presence, respectively, of a quencher at concentration [Q].
K
SV
is the Stern
−
Volmer constant, which quanti
fi
es the
dynamic quenching for a given position.
Steady-state anisotropy
r
was measured at an excitation
wavelength of 295 nm and in an emission range of 340
−
360
nm both with a slit width of 2 nm. The anisotropy was
determined according to eq 3 considering the instrumental
G
factor =
I
hv
/
I
hh
:
=−
+
r
IGII GI
()/(2)
vv
vh
vv
vh
(3)
Figure 1.
Stability of MscS tryptophan mutants. (A) Western blot of
E. coli
MscS Trp mutants in which 15
μ
g of membrane protein was loaded on a
4 to 12% sodium dodecyl sulfate
−
polyacrylamide gel electrophoresis gel and after development Western blotting with antibody speci
fi
c for the His
6
tag (see Experimental Procedures). (B) Membrane proteins (30
μ
g) were solubilized in PBS (pH 7.4) containing 1% DDM, 2.8 M urea, and 1 mM
EDTA and prepared for BN-PAGE as described in Experimental Procedures. Samples were separated on Novex 4 to 16% Bis-Tris gradient native
gels (Invitrogen) and proteins detected by Western blot as described for panel A. The positions of heptameric and monomeric MscS with associated
lipids and detergent are indicated.
Biochemistry
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DOI: 10.1021/acs.biochem.5b00294
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The
fi
rst and second subscripts indicate the position of the
excitation and emission polarizers (v for vertical and h for
horizontal), respectively.
■
RESULTS
Expression and Stability of the MscS Trp-Substituted
Channels.
With the single exception of A106W, expression
and integration into the membrane were observed (as detected
by Western blot for the His tag) for all the mutated MscS
channels (Figure 1A). Blue native (BN) PAGE analysis in
combination with Western blots is a convenient tool for
assessing the oligomeric state of MscS without the requirement
for full puri
fi
cation.
42
Preliminary work established that
detergent solubilization alone did not destabilize the protein.
Thus, the oligomeric state was assessed by incubation under
mildly destabilizing conditions (2.8 M urea and 1 mM EDTA).
Under these conditions, wild-type (WT) MscS, YFF, and 14 of
25 mutant proteins were essentially heptameric (Figure 1B).
Only seven of the mutant channels exhibited signi
fi
cant levels
of monomer after extraction in the presence of urea and EDTA
(G90W, T93W, V96W, V99W, V107W, G108W, and A110W).
Five other mutants exhibited some breakdown of the heptamer
in the presence of urea. Thus, the majority of the Trp mutants
display remarkable stability. This observation was supported by
analysis of a limited series of proteins by size exclusion
chromatography utilizing just those channels that were
subsequently studied by
fl
uorescence (Figure 2). For
fl
uorescence analysis, it is critical that the properties of the
Trp residues re
fl
ect the heptameric state rather than any
monomers that might arise after extraction from the membrane.
Five of 14 mutants exhibited signi
fi
cant levels of monomer in
the puri
fi
ed protein. Thus, WT protein eluted from the size
exclusion column predominantly as a single symmetrical peak
(Figure 2). We have previously reported that L109W is
unstable and this protein was used to validate the assay (Figure
2).
15
Unstable proteins, particularly R88W, I97W, A98W, and
G101W, exhibited increased amounts of the monomer and
decreased amounts of the heptamer. Some mutant proteins
exhibited a shift in the elution volume (e.g., N30W and
Q112W), and the elution pro
fi
le for others exhibited a small
peak on the leading shoulder for the heptameric protein
consistent with formation of higher-order oligomers (A94W,
A98W, and G113W). G90W, which was expressed well (Figure
1A) but very unstable in the presence of DDM and urea
(Figure 1B), could not be puri
fi
ed in signi
fi
cant quantities
(Figure 2). The small amount of pure protein obtained was
distributed between the heptamer and monomer, again
suggesting extreme instability caused by the Gly to Trp change
at this position.
Physiological Characterization of the Mutant Chan-
nels.
By three criteria [expression, stability after puri
fi
cation,
and BN-PAGE after DDM/urea extraction (Figures 1A,B and
2)], the majority of the Trp mutants are stable proteins suitable
for analysis. Previous studies have established that the
expression of MscS at the basal level driven by the Trc
promoter gives moderate protection to a strain deleted for
MscS, MscK, MscG (YbdG), and MscL (MJF612) and that
overexpression by induction with IPTG leads to complete
protection.
14
The survival assay works best as a predictor of loss
of either expression or function in mutated channel
proteins.
13,35
−
37
MscS channels that are poorly expressed or
have impaired function (or both) provide only limited
protection, and this is most easily seen when the channel
expression has not been induced.
37,43,44
The majority of the
Trp mutant channels failed to a
ff
ord protection when expressed
at this basal level, which is 8
−
10-fold below the expression level
normally seen for the fully induced WT protein (Figure S1 of
the Supporting Information and Figure 3A), but were
protective after induction with IPTG (Figure 3B). The
exception was T93W, which protected even when expressed
at a basal level (Figure 3A). Several mutant channels (R88W,
G90W, V91W, V96W, G108W, A110W, and Q112W)
exhibited complete loss of protective ability despite being
present in the cell membrane. Similar observations have been
made for some mutations a
ff
ecting the MscL channel.
45
The electrical signature of the Trp mutant MscS channels
was investigated using MscL as a reference point for the
activation threshold for the mutant channels.
38
The electrical
signatures of the mutants were position-speci
fi
c and could be
broken down into four principal groups with respect to the
stability of the openings, the conductance, and the pressure
sensitivity. Seven mutants (M47W, T93W, S95W, I97W,
G101W, G104W, and L111W) exhibited essentially normal
channel openings, but there were small di
ff
erences in the
P
L
:
P
S
ratio (Table 1 and Figure 4). However, the majority of the
mutants were found to exhibit either reduced conductance or
no conductance at all, and many also failed to form fully open
states, which precluded detailed analysis of their pressure
dependence (Table 1 and Figure S2 of the Supporting
Information). Insertion of Trp at positions V91, Q92, and
A94, which all map to the neck of the pore, generated channels
that were expressed (Figure 1A), exhibited some protection
Figure 2.
Size exclusion chromatographs of selected MscS tryptophan
mutants. Heptameric and monomeric MscS were separated on a
HiPrep Superdex 200 10/600 GL size exclusion column as described
in Experimental Procedures. A vertical dashed line indicates the
heptamer position for WT MscS.
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against downshock (Figure 3B), but failed to exhibit currents
despite the observation of MscL channels in the patches (Table
1). Three mutants (V96W, A103W, and V107W) opened to
the full extent, but the open state was unstable (Table 1).
A unique property was observed with V99W. At low
pressures (
P
L
:
P
S
= 1.8
−
2.4), its conductance was similar to
that of MscSYFF [
∼
1.1
±
0.01 nS (
n
= 5)]; however, the
channel
fl
ickered, and transitions to subconducting states were
frequently observed (Figure 5A), indicating that the open state
was unstable. However, if the pressure on the patch was
increased (
P
L
:
P
S
= 1.17
−
1.49), stable openings that were
∼
50%
larger than that of the WT [1.64
±
0.02 nS (
n
= 5) (Figure
5D)] were observed, and these exhibited a stability similar to
that of MscS (Figure 5A). In the closed crystal structure
[Protein Data Bank (PDB) entry 2OAU], V99 is exposed in the
crevice between TM1/TM2 and TM3a, which could
accommodate the increased volume of Trp (
∼
50% increase
relative to that of Val). In the open crystal structure (2VV5), it
is observed that the movement of TM3a outward from the pore
axis (to create the open pore) juxtaposes Val99 with Phe80,
which could generate a potential con
fl
ict between V99W and
F80 causing an unstable open state, but at higher pressures, the
two residues might form a
π
−
π
stack that stabilizes the more
conducting stable state (Figure 5B,C). Thus, we created a
V99W/F80A double mutant to introduce a compensating drop
in amino acid side-chain volume at position 80 and to remove
the potential for
π
−
π
stacking. The resulting channel exhibited
electrophysiological properties close to those of MscSYFF
(
P
L
:
P
S
= 1.54
±
0.06;
γ
∼
1.1 nS;
n
= 3) (Figure 5A, bottom
panel).
Polarity and Water Accessibility Detected by Fluo-
rescence Spectroscopy.
Tryptophan is a sensitive
fl
uoro-
phore that reports the physical properties of its environment.
46
We investigated the
fl
uorescence of detergent-solubilized MscS
channels with Trp at a number of positions between R88 and
G113. Positions in TM3a in which the native residue side chain
is oriented toward the pore were chosen, and in addition, three
mutants were investigated in the periplasmically exposed region
of the closed crystal structure of the channel (N30W, R88W,
and G90W). Two further mutants were created as reference
points: D213W is located on the surface of the cytosolic
domain and represents a residue completely exposed to the
bulk water phase, and M47W is in the middle of TM1. Size
exclusion chromatography revealed that several mutant forms
are less stable than WT MscS as discussed above. These were in
particular R88W, G90W, I97W, and A98W. If the protein were
a mix of heptamers and monomers in detergent, then this
would compromise the analysis of
fl
uorescence because the
data would represent the mix of proteins in multiple di
ff
erent
states. Anisotropy measurements using separate pools of
monomers and heptamers (see below) suggest that the
complex stays intact during measurement.
The peak position for each of the Trp residues was blue-
shifted relative to both the native Trp residues in the soluble
domain (
λ
max
= 334 nm for W240 in a mutant in which the
proximal Trp residue W251 had been mutated to Phe
20
) and
the surface-exposed Trp mutant, D213W (Table 2). The
emission maxima clustered together for most of the mutants
with the exceptions of A103W, G101W, G113W, and D213W
(Table 2 and Figure 6). The most blue-shifted, representing the
most hydrophobic environment, was A103W (
λ
max
= 310.5
±
0.4 nm), and this residue was least accessible to quenching by
acrylamide (Table 2 and Figure 6). This residue and T93W
were the only residues we studied by
fl
uorescence in TM3a that
are outward-facing (i.e., oriented away from the pore) in the
crystal structures. Overall, a correlation (
R
= 0.97) was seen
between
λ
max
and
K
SV
, which reports accessibility to the
aqueous quenching agent, acrylamide.
47
It is di
ffi
cult to predict
the precise positioning of the Trp side chain in the mutants, but
the
λ
max
and
K
SV
values (Table 2) are consistent with a
hydrophobic environment that is relatively inaccessible to
aqueous quenching agents (Figure 7), though in principle
stationary water could also cause a blue-shift of
λ
max
depending
on the position of the water dipole relative to the
fl
uorophore.
48
A surprising observation was that Trp residues
located at positions L105 and L109 reported signi
fi
cantly polar
environments and were moderately accessible to acrylamide
(Table 2 and Figure 7). Toward the cytosolic vestibule, the
acrylamide accessibility is increased at G113W, which is
consistent with the higher
λ
max
[329.7
±
1.3 nm (Table 2)].
Figure 3.
Protection against hypoosmotic shock. Transformants of MJF612 (lacking MscL, MscS, MscK, and YbdG) expressing individual MscS Trp
mutants were assessed for survival after a 0.5 M NaCl hypoosmotic shock. Mutants were either expressed as a result of the escape promoter activity
of the pTrc promoter (A)
14
or induced during growth (B; 0.3 mM IPTG for 30 min) immediately prior to downshock (see Experimental
Procedures). Data are means and the standard deviation of three independent cultures.
Biochemistry
Article
DOI: 10.1021/acs.biochem.5b00294
Biochemistry
2015, 54, 4519
−
4530
4523
Downloaded by CALIFORNIA INST OF TECHNOLOGY on August 27, 2015 | http://pubs.acs.org
Publication Date (Web): July 20, 2015 | doi: 10.1021/acs.biochem.5b00294