Role of protons in sugar binding to LacY
Irina Smirnovaa, Vladimir Kashoa, Junichi Sugiharaa, José Luis Vázquez-Ibara,1, and H. Ronald Kabacka,b,c,2
Departments ofaPhysiology andbMicrobiology, Immunology and Molecular Genetics andcMolecular Biology Institute, University of California, Los Angeles,
Contributed by H. Ronald Kaback, August 28, 2012 (sent for review July 28, 2012)
WT lactose permease of Escherichia coli (LacY) reconstituted into
proteoliposomes loaded with a pH-sensitive fluorophore exhibits
robust uphill H+translocation coupled with downhill lactose trans-
port. However, galactoside binding by mutants defective in lactose-
induced H+translocation is not accompanied by release of an H+on
the interior of the proteoliposomes. Because the pKavalue for ga-
lactoside bindingis∼10.5,protonation ofLacY likelyprecedessugar
binding at physiological pH. Consistently, purified WT LacY, as well
as the mutants, binds substrate at pH 7.5–8.5 in detergent, but no
change in ambient pH is observed, demonstrating directly that LacY
already is protonated when sugar binds. However, a kinetic isotope
effect (KIE) on the rate of binding is observed, indicating that deu-
terium substitution for protium affects an H+transfer reaction
within LacY that is associated with sugar binding. At neutral pH
or pD, both the rate of sugar dissociation (koff) and the forward rate
(kon) are slower in D2O than in H2O (KIE is ∼2), and, as a result, no
change in affinity (Kd) is observed. Alkaline conditions enhance the
effect of D2O on koff, the KIE increases to 3.6–4.0, and affinity for
sugar increases compared with H2O. In contrast, LacY mutants that
exhibit pH-independent high-affinity binding up to pH 11.0 (e.g.,
Glu325 → Gln) exhibit the same KIE (1.5–1.8) at neutral or alkaline
pH (pD). Proton inventory studies exhibit a linear relationship be-
tween koffand D2O concentration at neutral and alkaline pH, in-
dicating that internal transfer of a single H+is involved in the KIE.
deuterium isotope effect|membrane transporters|pH fluorophores|
most extensively studied membrane transport protein, with
several available crystal structures (1–4), mutagenesis of each
residue (5), and a wealth of biochemical/spectroscopic data re-
garding the conformational changes resulting in alternating ac-
cess, as well as the symport mechanism (reviewed in refs. 6 and
7). LacY is a member of the major facilitator superfamily (8, 9)
and catalyzes the coupled stoichiometric translocation of a ga-
lactoside and an H+(galactoside/H+symport) across the bacterial
cytoplasmic membrane, thereby transducing free energy stored in
an H+electrochemical gradient (Δμ∼H+) into a sugar-concentra-
tion gradient. Because transport is obligatorily coupled, downhill
transport of galactoside also drives uphill transport of H+with
generation of Δμ∼H+, the polarity of which is dictated by the di-
rection of the sugar-concentration gradient. The highly dynamic
nature of LacY (reviewed in ref. 7) is consistent with an alter-
nating access mechanism involving a global conformational
change in which sugar- and H+-binding sites are alternatively
exposed to either side of the membrane (reviewed in ref. 10).
In the kinetic model of transport proposed originally (11) and
supported by subsequent findings (reviewed in refs. 7 and 12),
protonation is required for sugar binding to LacY. Galactoside-
binding affinity decreases as pH is increased, with pKa∼10.5,
indicating that LacY is protonated over the physiological pH
range (13, 14). Pre–steady-state kinetic studies reveal that de-
creased affinity at alkaline pH is caused by a dramatic increase in
the rate of sugar dissociation (koff), whereas the forward rate
(kon) is independent of pH. Moreover, mutational analyses
demonstrate that the residues located in the H+-binding site
define the pKafor sugar binding and alter galactoside-binding
affinity (14). The amino acyl side chains involved in H+binding/
translocation (1–3) form a tightly interconnected H-bond/salt
he lactose permease of Escherichia coli (LacY) is arguably the
bridge cluster (Arg302, His322, Tyr236, and Asp240) in the
middle of the molecule with Glu325 and Lys319 within 5 Å on
opposite sides of the cluster (Fig. S1). Mutations in the core of
the H+-binding site decrease sugar affinity (14) and may cause
uncoordinated opening and closing of the cavities in LacY (15).
In contrast, replacing Glu325 (e.g., with an uncharged side
chain) blocks H+escape from the central core, and high-affinity
galactoside binding is maintained up to pH 11.0. The findings
suggest that the alkaline pKaobserved for sugar binding is not
related to a single amino acyl side chain but appears to be as-
sociated with a network of residues that include coordinated
water molecule(s) (14, 15).
Proton-coupled transporters use different mechanisms of sub-
strate and H+binding/release during transport. For example,
Schuldiner and coworkers (16–19) have shown elegantly that
binding of either substrate or H+to the homodimeric multidrug
antiporter EmrE involves two carboxyl residues (Glu14), one
from each monomer. The binding site can be occupied either by
one molecule of substrate or by two H+, but not by both simul-
taneously. Therefore, deprotonation is required for substrate
binding, and release of substrate is required for protonation.
Unlike EmrE, binding of both an H+and a galactoside as
cosubstrates is required for sugar/H+symport by LacY.
In this paper, we investigate the relationship between pro-
tonation of LacY and sugar binding with pH-sensitive fluores-
cence probes to define the order of galactoside and H+binding
directly. Purified WT LacY and mutants defective in H+trans-
location across the membrane either solubilized in N-dodecyl-
β-D-maltoside(DDM) or reconstituted into proteoliposomes
were tested for H+binding/release as a result of galactoside
binding. We show here that no H+binding is detected when
sugar is added to purified LacY in detergent, indicating that
protonation precedes substrate binding. However, D2O slows the
rate of sugar binding, and the effect of D2O on the kinetics of
sugar binding in combination with H+inventory studies reveals
that an internal H+transfer reaction is involved in sugar binding.
Sugar-Induced H+Influx into Proteoliposomes. The pH-sensitive
fluorophore 8-hydroxypyrene-trisulfonate (pyranine) was used to
test uphill H+translocation induced by downhill transport of
lactose (Fig. S2). Proteoliposomes containing WT LacY and
loaded with pyranine exhibit no change in fluorescence after
mixing with sucrose or buffer alone (Fig. 1A, traces 1 and 2,
respectively). However, addition of lactose generates a decrease
in fluorescence (trace 3), indicating acidification of the interior
of the proteoliposomes, and equilibrium is reached within 20 s
(t1/2is ∼3 s with 12 mM lactose). The same proteoliposomes
pretreated with N-ethylmaleimide, which blocks sugar binding
and transport by alkylating Cys148 (20), do not exhibit any
change in fluorescence after mixing with substrate (trace 4).
Author contributions: I.S., V.K., and H.R.K. designed research; I.S., V.K., J.S., and J.L.V.-I.
performed research; V.K. contributed new reagents/analytic tools; I.S., V.K., and H.R.K.
analyzed data; and I.S., V.K., and H.R.K. wrote the paper.
The authors declare no conflict of interest.
1Present address: Unitat de Biofísica, Facultat de Medicina, Universitat Autonoma de
Barcelona, 08193 Bellaterra, Barcelona. Spain.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 16, 2012
| vol. 109
| no. 42
Moreover, proteoliposomes reconstituted with mutant E325A,
which does not catalyze lactose/H+symport (21–26), exhibit no
acidification whatsoever after mixing with lactose (traces 5 and 6).
Binding of galactoside with the same proteoliposomes, as mea-
sured by Trp151→4-nitrophenyl-α-D-galactopyranoside (NPG)
FRET (27), is rapid, with similar rates for WT and E325A LacY
(Fig. 1B). Therefore, substrate appears to bind to LacY that al-
ready is protonated, because no detectable H+binding or release is
observed in proteoliposomes when overall symport does not occur.
Substrate Binds to Protonated LacY. Addition of a saturating con-
centration of β-D-galactopyranosyl-1-thio-β-D-galactopyranoside
(TDG) to purified LacY in unbuffered DDM solution containing
pyranine does not elicit any change in pH, which would be
expected if a stoichiometric amount of H+were bound or re-
leased (Fig. 2A). In contrast, with the antiporter EmrE as a
positive control (16, 17) under identical conditions, release of
∼0.7 mol of H+per mol of EmrE monomer is observed upon the
addition of tetraphenylphosphonium (TPP+) (Fig. 2B, black
trace), as shown previously by Schuldiner and coworkers (16–19).
Furthermore, shifting the pH to a more alkaline region also does
not result in a change in fluorescence upon the addition of TDG
to WT LacY (Fig. S3A), as monitored with 5-(and 6-) carbox-
ynaphthofluorescein (CNF), which has higher pKathan pyranine
(Fig. S2). However, under the same conditions, sugar binding is
detected readily by Trp151→NPG FRET (Fig. S3B).
Testing different LacY mutants at pH 8.3 with CNF as the pH
probe also shows no change in pH upon TDG binding (Fig. 3).
Notably, each mutant binds galactosides under these conditions,
although they exhibit different pKas for sugar-binding affinity
(Fig. S4) (14). The C154G mutant has the same pKa(∼10.5) as
WT LacY, whereas mutants R302K and Y236A display pKas of
8.0–8.4, and E325A exhibits high sugar-binding affinity that is
independent of pH up to pH 11. Clearly, therefore, with neither
WT LacY nor any of the mutants tested does binding of galac-
toside elicit any change in ambient pH.
Deuterium Kinetic Isotope Effects.Rates of sugar binding to C154G
LacY were measured in H2O or D2O at a pH (pD) of 7.0 (Fig.
4). Binding rates (kobs) measured directly by Trp151→NPG
FRET depend linearly on NPG concentration and are distinctly
slower when D2O is substituted for H2O (Fig. 4 A and B). The
finding is not caused by an effect of viscosity on sugar binding,
because 10% glycerol, which increases viscosity more than 98%
D2O (28), does not change the binding rate significantly (Fig.
4B). In addition, prolonged preincubation of NPG in D2O does
not change the binding kinetics (Fig. S5), thus excluding an
effect of sugar deuteration on binding rates. koffmeasured by
displacement of bound NPG with an excess of TDG also is sig-
nificantly slower in D2O than in water (Fig. 4C). However, Kd
values are the same in H2O and D2O (Fig. 4D). Therefore, D2O
affects both konand koffequally. Estimated from direct binding,
konvalues are 5.2 ± 0.1 and 2.4 ± 0.1 μM s−1in H2O and D2O,
respectively (Fig. 4B). The koffvalues calculated from displace-
ment experiments (Fig. 4C) are 70.3 ± 1.4 and 39.7 ± 1.2 s−1in
H2O and D2O, respectively. Thus, the deuterium kinetic isotope
effects (KIE) calculated from these data are ∼2 at neutral pH
(pD) for C154G LacY.
WT LacY (four upper traces) or E325A mutant (two lower traces) was recon-
stituted in proteoliposomes, loaded with 0.6 mM pyranine, and mixed with
sucrose (trace 1), lactose (traces 3, 4, and 6), or buffer only (traces 2 and 5). To
block sugar binding, proteoliposomes with WT LacY were preincubated with
10 mM N-ethylmaleimide for 10 min before mixing with lactose (trace 4). The
sugar concentration after mixing was 12 mM, and the final protein concen-
tration was ∼0.5 μM in 5 mM KPi/100 mM KCl (pH 7.5) with 1 μM valinomycin.
The excitation wavelength for pyranine was 450 nm, and emitted light was
to WT LacY and E325A mutant by Trp151→NPG FRET. Stopped-flow traces of
the decrease in Trp fluorescence were recorded after mixing proteoliposomes
with NPG (100 μM, final concentration). Buffer content and proteoliposomes
(without pyranine) are the same as in A. The excitation wavelength was 295
nm, and emitted light was collected with a long-pass filter at 320 nm. Single-
exponential fits are shown as black lines with estimated rates of 16.3 ± 0.6 s−1
and 8.6 ± 0.5 s−1for WT LacY and mutant E325A, respectively.
Influx of H+into proteoliposomes by symport with lactose. Stopped-
detergent. Fluorescence of pyranine (40 nM) was recorded continuously at
excitation and emission wavelengths of 450 and 510 nm, respectively, in
unbuffered solution containing a given protein (black traces, 2) or solution
with no protein (gray traces, 1). Additions are indicated by arrows. (A)
Changes in pyranine fluorescence after the addition of TDG (5 mM) to 100
mM NaCl/0.02% DDM without protein (gray trace) or containing 10 μM LacY
(black trace). The starting point corresponds to pH 7.4. Additions of 10 μM
HCl or NaOH were made to quantify the shift in fluorescence intensity
caused by the change in H+concentration. (B) Changes in pyranine fluo-
rescence after the addition of 40 μM TPP+to 100 mM NaCl/0.08% DDM
solution without protein (gray trace), or with 4 μM EmrE (60 μg/mL) (black
trace). The starting point corresponds to pH 7.2. Additions of 5 μM HCl or
NaOH were made to quantify the shift in fluorescence intensity caused by
the change in H+concentration. Estimated proton release triggered by TPP+
binding is 0.7 mol/mol of EmrE monomer.
Changes in pH induced by substrate binding to LacY or EmrE in
| www.pnas.org/cgi/doi/10.1073/pnas.1214890109 Smirnova et al.
The pKaof ∼10.5 for sugar-binding affinity measured for LacY
resultsfrom a dramatic increase of koffat alkaline pH,whereas kon
is independent of pH (13, 14). Therefore, displacement rates and
NPG-binding affinities were measured in H2O and D2O at a pH
(pD) of 10 (Fig. 5). Stopped-flow rates of NPG displacement by
TDG (koff) measured in H2O are significantly faster at pH 10 than
at neutral pH (compare gray traces in Figs. 4C and 5A). An effect
of D2O on koffis observed at both pH values but is much more
of 3.6–4.0 (Figs. 4C, 5A, and 6 C and D and Table 1). The Kdfor
NPG is determined from the amplitude of the stopped-flow traces
at different NPG concentrations (Fig. 5B). Sugar-binding affinity
decreases greatly in aqueous solution at pH 10, (the Kdincreases
from 16 μM at pH 7 to 240 μM at pH 10). Remarkably, the de-
crease in affinity (i.e., increase in Kd) is significantly smaller when
pH dependence of koffdescribed for C154G LacY also is observed
for WT LacY (Fig. 6 A and B). The KIE calculated from rates of
NPG displacement (koff) increases from 1.5 at pH (pD) 7.0 to 3.6
at pH (pD) 10.0 (Table 1).
A KIE also is observed with three LacY mutants that have
significantly different pKas for sugar-binding affinity. Mutants
E325Q and K319L bind sugar with high affinity up to pH 11.0,
whereas K319R binds sugar in a manner similar to WT LacY
with pKa∼10.5 (14). All the mutants exhibit KIEs at neutral pH
similar to WT LacY, with values ranging from 1.5–2.0 (Fig. 6,
Left and Table 1). Notably, at pH 10.0 the KIE for mutants
E325Q and K319L remains independent of pH (pD) (Fig. 6,
Right and Table 1), whereas the KIE for mutant C154G and
K319R (4.0 and 3.6, respectively) is very similar to that obtained
for WT LacY.
Because the H+concentration in the surrounding medium does
not change with galactoside binding to LacY (Figs. 2 and 3 and
Fig. S3), and a D2O effect is observed at neutral pH (KIE 1.5–2.0)
and at alkaline pH (KIE 3.6–4.0), the possibility arises that more
than one H+is involved with the binding reaction. Therefore, the
proton inventory technique was used (29–32), and NPG dis-
placement rates (koff) were determined at neutral and alkaline
conditions at increasingconcentrationsof D2O (Fig. 7).Asshown,
WT LacY and the four mutants described above exhibit a linear
relationship between koffand D2O concentration at pH (pD) 7.0
or 10.0. The data suggest that a single H+is involved in the KIE.
The slope of the line is greater at pH (pD) 10.0 than at pH (pD)
7.0 for WT LacY, C154G, and K319R, corresponding to higher
KIEs for these proteins under alkaline conditions (Table 1) and
reflecting pH-dependent sugar binding (14). In contrast, mutants
E325Q and K319L display similar KIEs at both pH values, in
agreement with pH-independent sugar binding, and similar D2O
effects on koffat pH (pD) 7.0 or 10.0 (Fig. 7 E, F, I, and K).
As expected, downhill transport of sugar by LacY is obligatorily
coupled to H+translocation and thereby causes acidification of
the interior milieu of proteoliposomes. Alkylation of Cys148 in
the vicinity of a sugar-binding site prevents sugar binding and
completely blocks lactose/H+symport. Also, no acidification is
observed with mutant E325A, which binds galactoside normally
detected by CNF fluorescence. Fluorescence of CNF (1 μM) was recorded
continuously at excitation and emission wavelengths of 600 and 665 nm,
respectively, in unbuffered solution (100 mM NaCl/0.02% DDM) containing
protein. The starting pH was 8.1–8.3. Additions of TDG (5 mM), NaOH, and
HCl are indicated by arrows. Protein concentrations were 10 μM C154G (A),
6 μM E325A (B), 10 μM R302K (C), and 10 μM Y236A (D). All proteins bind
galactosides well, as determined by Trp151→NPG FRET (Fig. S4 E–H).
Effect of substrate binding to LacY mutants on pH in solution
NPG binding to C154G LacY was measured by stopped flow as Trp151→NPG
FRET with excitation at 295 nm using an emission long-pass filter at 320 nm.
The final protein concentration was 0.5 μM. The buffer (25 mM NaPi/100 mM
NaCl/0.02% DDM) was prepared in H2O, 10% glycerol, or D2O with pH (pD)
adjusted to 7.0 (pD = pH + 0.4). (A)Stopped-flow traces recorded after mixing
of protein with NPG at the given final concentrations in H2O (gray traces) or
D2O (black traces). Single-exponential fits are shown as solid black lines. (B)
Dependence of rates measured as shown in A in H2O (gray circles), 10%
glycerol (gray triangles), or D2O (black circles) on NPG concentration. The kon
values estimated from linear fits are 5.2 ± 0.1, 4.1 ± 0.1, and 2.4 ± 0.1 μM s−1
inH2O, 10% glycerol, andD2O, respectively. (C) Stopped-flowtraces recorded
after mixing 15 mM TDG (final concentration) and LacY preincubated with
NPG at given concentrations in H2O (gray traces) or D2O (black traces). Single-
exponential fits are shown as solid black lines. The estimated koffvalues are
70.3 ± 1.4 and 39.7 ± 1.2 s−1in H2O and D2O, respectively. The vertical dashed
line indicates dead-time of the instrument (1.5 ms). (D) NPG-binding affinity
in H2O or D2O determined from the concentration dependence of the fluo-
rescence change estimated from single-exponential fits as shown in C. The
relative change in fluorescence at each NPG concentration is calculated as the
percentage of the final fluorescence level after displacement of NPG by an
excess of TDG. Data obtained in H2O and D2O are presented as gray and back
symbols, respectively. Kdvalues estimated from hyperbolic fits are 15.9 ± 0.8
and 18.8 ± 2.7 μM in H2O and D2O, respectively.
Effect of D2O on sugar binding to LacY. Pre–steady-state kinetics of
Smirnova et al. PNAS
| October 16, 2012
| vol. 109
| no. 42
but is defective in reactions that involve net H+translocation (21–
26). Therefore, binding of sugar alone is not followed by binding
or release of a stoichiometric H+on the cytoplasmic side of LacY
inside the proteoliposomes. Thus, protonation or deprotonation as
the direct result of galactoside binding was investigated with pu-
rified proteins solubilized in DDM at high protein concentrations.
Nochange inambientpHisobserveduponsugar binding to WT
LacY or mutants C154G, E325A, R302K, and Y236A under
conditions in which ligand binding to EmrE results in dissociation
of close to a single H+/mol of EmrE monomer, as shown by
Schuldiner and colleagues (16, 17). Notably, mutants Y236A or
R302K in the core of the H+-binding site exhibit markedly de-
creased affinity (i.e., increased Kd) and shifted pH dependence of
sugar-binding affinity (pKa= 8.0–8.4) compared with WT LacY
binding with these mutants, thereby suggesting that LacY must
already be protonated before sugar binds. Similarly, no change in
high-affinity NPG binding is independent of pH up to pH 11.0.
Protonation is required for sugar binding under physiological
conditions (13), and the results presented here support and ex-
tend this interpretation. Mutational analysis reveals that in-
teraction of H+with several amino acid residues, which form
a tightly interconnected H-bond/salt bridge cluster (Fig. S1B), is
essential for high-affinity sugar binding (14). Substitution of
deuterium for protium should affect proton transfer within the
H+-binding site and therefore may alter reaction rates that de-
pend on protonation/deprotonation. Hence, the effect of D2O
on the kinetics of sugar binding to WT LacY and mutants was
investigated. Previously it was shown that D2O decreases the rate
of downhill lactose transport with no effect on the rate of Δμ∼H+
-driven uphill transport or equilibrium exchange (33). These and
other findings (11, 25, 26, 34) support the conclusion that the
rate-limiting step for downhill lactose/H+symport is deproto-
nation and that this step no longer is rate-limiting when there is
a driving force on the H+in the form of Δμ∼H+ or when sugar
transfer across the membrane does not involve H+translocation
measurements of Trp151→NPG FRET at an excitation wavelength of 295 nm
with an emission long-pass filter of 320 nm. The final protein concentration
was0.5 μM in 25mMCAPS/100 mMNaCl/0.02%DDMata pH (pD)of 10.0 (pD =
pH + 0.4). (A) Stopped-flow traces recorded after mixing of 15 mM TDG (final
concentration) and protein preincubated with NPG at given concentrations in
H2O (gray traces) or D2O (black traces). Single-exponential fits are shown as
solid black lines. Theestimated koffvalues are680± 101 and 188± 28s−1inH2O
or D2O, respectively. The dashed broken line indicates dead time of the in-
strument (1.2 ms). (B) NPG-binding affinity in H2O or D2O is determined from
the dependence of the fluorescence changes on NPG concentration as de-
scribed in Fig. 4D. The relative change in fluorescence at each NPG concentra-
tion was calculated as a percentage of the final fluorescence level after
displacement of NPG by an excess of TDG. Data obtained in H2O and D2O are
presented as gray and black triangles, respectively. Kdvalues estimated from
hyperbolic fits are 240 ± 80 and 76 ± 5 μM in H2O and D2O, respectively.
Effect of D2O on sugar binding to LacY at pH 10.0. Pre–steady-state
kaline pH (pD). Rates of displacement of bound NPG by an excess of TDG
were determined by measuring Trp151→NPG FRET at excitation and emis-
sion wavelengths of 295 and 330 nm, respectively. Stopped-flow rates were
measured in H2O (gray traces) and D2O (black traces). Final concentrations
were 1 μM protein, 100 μM NPG, and 6 mM TDG. The buffers (25 mM NaPi
or CAPS with 100 mM NaCl/0.02% DDM) were prepared in H2O or D2O with
pH (pD) adjusted to 7.0 or 10.0 (pD = pH + 0.4). The vertical dashed line
indicates dead-time of the instrument (2.7 ms). The rates estimated from
single-exponential fits are given. Data are shown for WT LacY (A and B) and
mutants C154G (C and D), E325Q (E and F), K319R (G and H), and K319L (I
and K). Panels on the left show traces recorded at pH (pD) 7.0, and panels
on the right show traces for pH (pD) 10. The estimated KIE for each protein
is shown in Table 1.
Effect of D2O on the NPG dissociation rate (koff) at neutral or al-
| www.pnas.org/cgi/doi/10.1073/pnas.1214890109 Smirnova et al.
(i.e., equilibrium exchange or counterflow). Here we investigated
the effect of D2O on the sugar-binding step under pre–steady-
state conditions using Trp→NPG FRET. Stopped-flow rates of
sugar binding (kon) and displacement (koff) were measured at pH
(pD) 7.0 and 10.0. At neutral pH, the effect of D2O on NPG
binding and displacement rates is comparable (KIE 1.5–1.9).
Notably, sugar-binding affinity at neutral pH is the same in either
H2O or D2O (Kd= 16–19 μM) as a result of concomitant changes
in both koffand kon. It is debatable whether noncovalent inter-
actions such as ligand binding result in a true KIE (32, 35, 36).
Thus, it should be emphasized that our observations likely rep-
resent an effect of deuterium on H+transfer within the H+
-binding site of LacY that alters sugar-binding rates and not
a direct effect of sugar binding itself. Moreover, this H+transfer
within LacY may correspond to the weak electrogenic reaction
triggered by sugar binding to the protein, one of two electrogenic
steps in the overall transport cycle of LacY revealed by solid-
supported membrane-based electrophysiology (25, 26).
The enhanced affinity for NPG observed in D2O at pD 10
results specifically from an increased KIE on koff. The measured
KIE is 3.6–4.0 for WT LacY and the mutants that exhibit a pKa
of ∼10.5 (Table 1). The much slower koff observed in D2O
compared with H2O could be explained either by an increase in
the true KIE at alkaline pH or by a general pKashift toward the
alkaline region (Fig. S6). Modeling pH dependencies in D2O
demonstrates that it is not possible to discriminate between the
alternatives experimentally (Fig. S6; compare curves 2 and 3 at
The requirement for protonation preceding sugar binding by
LacY and a significant KIE in D2O motivated an attempt to
assess the number of H+involved by using the proton inventory
technique (29–32). When the dependence of koffon D2O con-
centration is measured with purified WT LacY and four mutants
at pH (pD) 7.0 or 10.0, no significant deviation from linearity is
observed (Fig. 7). Thus, it is likely that a single proton is re-
sponsible for deuterium isotope effect on sugar-binding rates.
Materials and Methods
Materials. Oligonucleotides were synthesized by Integrated DNA Technolo-
gies, Inc. Restriction enzymes were purchased from New England Biolabs.
The QuikChange II kit was purchased from Stratagene. TDG was obtained
from Carbosynth Limited. NPG, 3-(cyclohexylamino)-1-propanesulfonic acid
(CAPS), and pyranine were obtained from Sigma-Aldrich. Talon Superflow
Resin was purchased from BD Clontech. CNF was from Life Technologies.
DDM and octyl-β-D-glucoside (OG) were obtained from Affymetrix. Synthetic
1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmi-
toyl-2-oleyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) were from Avanti
Polar Lipids, Inc. D2O (>99% vol/vol) was obtained from Spectra Stable Iso-
topes. All other materials were of reagent grade and were obtained from
commercial sources. Purified EmrE was a generous gift from Shimon Schuldiner
(Hebrew University of Jerusalem, Jerusalem, Israel).
Construction of Mutants, Purification of LacY, and Reconstitution into
Proteoliposomes. Construction of mutants, expression in E. coli, and purifi-
cation of LacY were performed as described (37). All mutants contained a C-
terminal 6-His tag that was used for affinity purification by cobalt affinity
chromatography on Talon resin. Purified proteins (10–15 mg/mL) in 50 mM
Table 1.Effect of pH on KIEs for WT LacY and mutants
LacY (pKa) pH or pD
WT (10.5)7 51/33
Values for koffwere determined as described in Fig. 6. Estimated pKa
values for sugar-binding affinity are from ref. 14.
were measured as described in Fig. 6 at given concentrations of D2O. The
ratio of displacement rate measured in the mixture of H2O + D2O to the
displacement rate measured in H2O is plotted versus the D2O concentration.
Data are presented for WT LacY (A and B) and mutants C154G (C and D),
E325Q (E and F), K319R (G and H), and K319L (I and K). Panels on the left
(filled symbols) show data for pH (pD) 7.0; panels on the right (open symbols)
show data for pH (pD) 10.0.
Proton inventory at neutral and alkaline pH (pD). Stopped-flow rates
Smirnova et al. PNAS
| October 16, 2012
| vol. 109
| no. 42
NaPi/0.02% DDM (pH 7.5) were frozen in liquid nitrogen and stored at Download full-text
−80 °C until use. Reconstitution of LacY into proteoliposomes was carried
out with synthetic phospholipids (POPE/POPG ratio 3:1) by using the dilution
method (38). Briefly, purified LacY in 0.02% DDM was mixed with phos-
pholipids (40 mg/mL) dissolved in 1.2% OG maintaining a lipid-to-protein
ratio of 5 (wt/wt). The mixture was kept on ice for 20 min and then was
diluted quickly in 50 mM NaPi(pH 7.5) so that final concentration of OG
decreased to ∼0.01% (critical micelle concentration = 0.53%). After 5 min
stirring at room temperature, the proteoliposomes were collected by cen-
trifugation for 1 h at 100,000 × g and were subjected to two cycles of freeze-
thaw/sonication before use. Loading of proteoliposomes with pyranine was
performed by sonication (39). Proteoliposomes (0.25 mL with protein con-
centration 2 mg/mL) were mixed with 2.25 mL (111 mM) KCl containing
0.6 mM pyranine, were sonicated in the water bath, and were subjected to
two cycles of freeze-thaw/sonication. Excess pyranine was removed by gel
filtration on a G-25 Sephadex column (1.5 × 12.0 cm) equilibrated with 5 mM
KPi/100 mM KCl (pH 7.5). Pyranine-loaded proteoliposomes completely
separated from the free fluorophore were collected in 3.5–4 mL.
Fluorescence Measurements. For experiments in which pH was monitored,
proteins were equilibrated with unbuffered solutions using Amicon Ultra
concentrators with a cutoff of 50 kDa for LacY and 10 kDa for EmrE. The
starting pH level of solutions containing an indicated pH-sensitive fluores-
cent probe and 4–10 μM protein was measured with a pH-meter and ad-
justed by the addition of small aliquots of diluted NaOH or HCl. Binding/
release of H+was quantified by measuring fluorescence intensity in a pH
range around the pKaof the fluorophore. Steady-state fluorescence was
monitored at room temperature on a SPEX Fluorolog 3 spectrofluorometer
(Horiba) in a 2.5-mL cuvettete with constant stirring. Typically additions of
1–5 μL were made during the time course of fluorescence monitoring.
Stopped-flow measurements were performed at 25 °C on a stopped-flow
device (dead-time 2.7 ms) using an SLM-Aminco 8100 spectrofluorimeter
(SLM Instruments) modified by OLIS, Inc. as described (27). Alternatively, an
SFM-300 rapid kinetic system equipped with TC-50/10 cuvettete (dead-time
1.2–1.5 ms) with an MOS-450 spectrofluorimeter (Bio-Logic USA, LLC) was
used as described (40). Typically, 10–15 stopped-flow traces were recorded for
each data point, averaged, and fitted with an exponential equation using the
built-in Bio-Kine32 software package or by using Sigmaplot 10 (Systat Soft-
ware Inc). All concentrations given are final concentrations after mixing.
D2O/H2O Mixtures. Stock solutions for the experiments were made either in
pure water or D2O (98% final concentration). The pH was adjusted by the
addition of small aliquots of either HCl or NaOH with a pH meter (pD = pH +
0.4). Given ratios of D2O/H2O-based solutions were prepared for the proton
inventory experiments by mixing appropriate volumes of stock solutions.
Small aliquots of concentrated proteins or sugars were diluted in the same
buffers and used immediately for stopped-flow measurements.
ACKNOWLEDGMENTS. We thank Shimon Schuldiner for providing samples
of purified EmrE and Klaus Fendler for critically reading the manuscript and
for stimulating discussions. M. Gregor Madej was instrumental in suggesting
the proton inventory studies. This work was supported by National Institutes
of Health Grants DK051131, DK069463, and GM073210 and by National
Science Foundation Grant MCB-1129551 (to H.R.K.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1214890109Smirnova et al.