Figure S1.
Detection levels of MscS in total membrane preparations. The plasmid pTrcYH
6
expressing C-terminally His-tagged
MscS was transformed into MJF641 (
Δ
7) and total membrane proteins were harvested from cells grown in a minimal citrate-
phosphate medium pH7. An overnight culture was grown with 0.04% glucose and in the next day supplemented with 0.2% glucose
until OD
650
= 0.4. A subsequent 100x dilution was performed and growth continued until cells reached OD
650
= 0.4. The culture was
split and cells one portion of cells was induced for expression of MscS by addition of 1 mM IPTG for 30 min prior to harvest. No
growth inhibition resulted from expression and the two portions of culture (induced and uninduced) were harvested in parallel. Total
membrane proteins were prepared as previously described in EXPERIMENTAL PROCEDURES. Lane 1: 15
μ
g of uninduced MscS.
Lanes 2 to 8: 2-fold serial dilutions (15
μ
g to 0.23
μ
g protein) of IPTG-induced samples in PBS. Volumes loaded per lane were kept
constant (15
μ
l). Samples were separated by SDS/PAGE 12% acrylamide gels and the MscS protein recognized by anti-His HRP
conjugated antibody and the ECL substrate Supersignal® West Dura Extended Duration. Figure shown is from a single gel/blot/film
after 1 min exposure time. The approximate mass of MscS was 20 kDa.
1 2 3 4 5 6 7 8
Imemb: 50 pA, Pressure: 340 mmHg
0.2 s
N30W
Imemb: 50 pA, Pressure: 340 mmHg
0.2 s
M47W
Imemb: 50 pA, Pressure: 350 mmHg
0.2 s
R88W
Imemb: 50 pA, Pressure: 350 mmHg
0.2 s
S95W
Imemb: 50 pA, Pressure: 350 mmHg
0.2 s
L100W
Imemb: 50 pA, Pressure: 11000 mmHg
0.2 s
G101W
Imemb: 50 pA, Pressure: 11000 mmHg
0.2 s
A102W
50
pA
0.2 s
Imemb: 50 pA, Pressure: 11000 mmHg
0.2 s
A103W
Imemb: 50 pA, Pressure: 350 mmHg
0.2 s
G104W
Imemb: 50 pA, Pressure: 11000 mmHg
0.2 s
V107W
Imemb: 50 pA, Pressure: 340 mmHg
0.2 s
L109W
Imemb: 50 pA, Pressure: 11000 mmHg
0.2 s
L111W
Imemb: 50 pA, Pressure: 340 mmHg
0.2 s
Q112W
Imemb: 50 pA, Pressure: 350 mmHg
0.2 s
G113W
Figure S2. The figure shows typical traces for the
Trp
mutants created in this study but not shown in the main
body of the paper. Details as described in Experimental
Procedures.
Figure
S3: Steady
state
anisotropy
of tryptophan
plotted
against
the
distance.
Distances
of side
chain
C
β
-atoms
between
the
same
residue
on neighbouring
subunits
were
obtained
from
the
closed
(2OAU
) and
open
(2VV
5) crystal
structures
and
plotted
against
the
anisotropy
r. The
data
(residue
number
in blue)
were
fitted
assuming
homoFRET
between
the
tryptophan
residues
(red
equation),
where
R
0
is the
Förster
distance
while
A and
B are
scaling
factors
. The
goodness
of fits
are
represented
by the
adjusted
R
2
values
and
the
fitted
parameters
are
given
with
the
standard
errors
of fitting
. It can
be
seen
that
the
fit for
the
closed
structure
(A)
is better
than
for
the
open
structure
(B).
Additional
fit are
shown
for
the
open
structure
where
the
green
points
were
not
considered
(C to E).
A
6
8
10
12
14
16
18
20
22
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
anisotropy
distance C
β
-C
β
(Angstrom)
푟푟
=
퐴퐴
1+
(
푑푑푑푑푑푑푑푑
푅푅
0
)
6
+B
closed structure: 2OAU
Adj. R
2
= 0.87
R
0
= 7.4
±1.3 Å
A = -
0.124±
0.051
B = 0.186±
0.007
30
88
90
92
93
94
97
98
101
103
105
109
112
113
6
8
10
12
14
16
18
20
22
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
anisotropy r
distance C
β
-C
β
(Angstrom)
open structure: 2VV5
Adj. R
2
= -0.04
R
0
= 10.7±
5.7 Å
A = -
0.05±
0.06
B = 0.176±
0.024
30
88
90
92
93
94
97
98
101
103
105
109
112
113
B
6
8
10
12
14
16
18
20
22
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
anisotropy
distance C
β
-C
β
(Angstrom)
D
open structure: 2VV5
Adj. R
2
= 0.36
R
0
= 12.0±
2.9
Å
A = -
0.07±
0.03
B = 0.181±
0.019
30
88
90
92
93
94
97
98
101
103
105
109
112
113
6
8
10
12
14
16
18
20
22
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
anisotropy
distance C
β
-C
β
(Angstrom)
C
open structure: 2VV5
Adj. R
2
= 0.25
R
0
= 7.3
±2.5
Å
A = -
0.12±
0.17
B = 0.183±
0.008
30
88
90
92
93
94
97
98
101
103
105
109
112
113
6
8
10
12
14
16
18
20
22
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
anisotropy
distance C
β
-C
β
(Angstrom)
E
open structure: 2VV5
Adj. R
2
= 0.51
R
0
= 8.9
±2.4
Å
A = -
0.08±
0.06
B = 0.179±
0.007
30
88
90
92
93
94
97
98
101
103
105
109
112
113
Figure
S4: Blue
native
PAGE
of selected
MscS
mutants
.
(A)
MscS
samples
of selected
mutants
were
directly
taken
from
peak
fraction
after
SEC
chromatography
(right
side
of gel),
For
the
mutant
T93W,
samples
for
the
heptameric
complex
as well
as dissociated
monomers
were
taken
(see
B). MscS
was
reconstituted
and
fluorescence
spectra
were
measured
.
Afterwards
the
samples
were
concentrated
with
Microcon
YM
-10
spin
filters
(Millipore),
re-
solubilised
in DDM
and
taken
as samples
for
the
BN
gel
(left
side
of the
gel)
which
showed
still
heptameric
complex
composition
. A NativePAGE
Novex
4-16% gel
(LifeTechnologies
) was
used
as described
by the
manufacturer
and
further
coomassie
-stained
afterwards
(GelCode
Blue,
Thermo
Scientific)
. MscS
complex
masses
can
be estimated
with
the
help
of the
soluble
marker
proteins
(first
lane
; NativeMark
, Life
Technologies)
and
a correction
factor
of 1.8 for
bound
dye
and
detergent
(Rasmussen
et al., 2007
; Heuberger
et al., 2002
). (B)
MscS
YFF
T93W was
separated
on
a Superdex
200
10/300
GL
column
.
T93Whep
A94W
G101W
G113W
T93Whep
T93Wmono
A94W
G101W
G113W
reconstituted solubilised in DDM
kDa
1048
720
480
242
146
66
heptamer
monomer
2*7
10
15
20
25
30
0
20
40
60
80
100
120
140
160
A
280nm
Elution volume (ml)
T93W
monomer
heptamer
A
B
Figure S5. Tryptophan scanning mutagenesis of
E. coli
MscS
.
The image (created in
Pymol
) illustrates the proximity of N30
and R88 on TM1 and TM2, respecFvely.
N30
R88
TM1
TM2
TM3a