5 Applications

5 Applications
5.1 Enzymology
• Single-molecule turnovers
• Mechanism of catalysis: Conformational dynamics
• Correlate with activity
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme
5.1.1 Activity
• Goal: find heterogeneities
• static: heterogeneities amongst molecules
• dynamic: heterogeneity of one molecule over time
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme, 5.1.1 Aktivität
2
2
2
Cholesterol-Oxidase
Downl
COx (23) shows that the FAD is noncoffusion (11–13),
valently and tightly bound to the center of the
ctral fluctuation
protein and is surrounded by a hydrophobic
(16 ), and photobinding pocket for cholesterol, which is othve been demonerwise filled with 14 water molecules.
is the real-time
Co factorAFAD
serves electron transfer
fluorescence image of single COx moltions of biomolecules in their oxidized form (Fig. 1A) was
of a few motor
turnover
/ reduction
taken
with anoxidation
inverted fluorescence
micro-of co faktor,
monitored During
in real
scope
by raster-scanning
the /sample
with a
eported here,
we
change
between
fluorescent
non fluorescent
fixed He-Cd laser (442 nm, LiCONiX) focus
rs of single flanitoring the fluosites. Statistical
cs at the singleights into enzy-
•
•
Northwest National
ronmental MolecuWA 99352, USA. L.
ty, Department of
, USA.
d be addressed. E-
Scheme 1.
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme, 5.1.1 Aktivität
SM - Turnover
ble-averaged enzymatic assays for COx (Sigma) in agarose gels yielded turnover rates
similar to those in aqueous solutions (26, 27).
Unlike the enzyme molecules, small substrate
molecules (cholesterol and oxygen) undergo
essentially free translational diffusion within
the gel.
With excess amounts of cholesterol (0.2
is independent of excitation inten
the blinking is due only to the
reactions in the ground electronic s
than photoinduced phenomena. (ii
eraged turnover rates of the traje
the same as the ensemble-average
rates under similar conditions (26,
The length of the trajectories is
• Every spot one COx-molecule, transient intensity
shows turnovers
A
80 ct
0
2
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme, 5.1.1 Aktivität
4
6
8
!m
State analysis
• Ping-Pong-mechanism
Downloaded from w
ide interfertremely
infrequent
blinking
was by
seen
during
decrease
with
timea because
substrate
(cholesmolcannot
usually
be
determined
ensemblezyme
(E)
involves
flavin
adenine
dinuclean electron
long averaged
trajectories
for only a Moreover,
few, but not
all,
terol
and oxygen)
concentrations
were in
orders
ster
g mode, and
measurements.
stochasotide (FAD),
which is
naturally fluorescent
(21 ns). The
COx tic
molecules,
we attribute toproperty
impuof
magnitude
the enery
trajectorieswhich
of a single-molecule
its oxidized
formhigher
but not than
in its those
reducedofform.
eral systemrity ofcan
substrate
molecules
a low-quantumzyme.
Theis long
trajectories
detailed
ited
be recorded
in real or
time,
containing deThe FAD
first reduced
by apermit
cholesterol
g the analytdynamical
information
extractable
molecule toanalyses.
FADH2, and is then oxidized by
yield tailed
photoinduced
process
(1, 4, 17).
(ii) In
statistical
COx
We measured
andards with
yielding
crystal structure
of
O2, The
through statistical
analyses.
the presence
of cholesterol,
theSingle-molecule
turnover rate
mostHobvious
of the turnover
em2O2. The feature
(spinel from
COx (23) in
shows
theitsFAD
is noncotrajectories of
is independent
oftranslational
excitation diffusion
intensity;(11–13),
thus,
trajectory
Fig. that
1B is
stochastic
nature.
Sigpan), augite
valently
and tightly boundbasis,
to the center
of the of a
rotational isdiffusion
(14),tospectral
fluctuation
and
synthetic
the blinking
due konly
the enzymatic
On
a single-molecule
the event
ates
1
18O/16O on
(15), conformational motion (16 ), and photo- k2protein and is surrounded by a hydrophobic
reactions
in the
state rather
chemical
reaction
takes
on the subpico27).
tandard was
E chemical
FAD
+ground
S (17,electronic
E haveFAD
•S !
E pocket
FADH
P which
2 +place
binding
for
cholesterol,
is othchanges
18)
been
demonfrate
O isotopic
than photoinduced
phenomena.
avsecond
timewith
scale
and cannot
be time rek 1interest is(iii)
erwise filled
14 water
molecules.
strated. Of particular
the The
real-time
s than 5 per
eraged
turnover ofrates
of the
are
solved
here.0 However,
time
needed
rgo
k01trajectories
s. Therefore,
A fluorescence
image ofthe
single
COx
mol- for
observation
chemical
reactions
of biomolk
2
isotope
the
same
as
the
ensemble-averaged
turnover
diffusion,
thermal
or
both
before
hin ratio
ecules
in
their
form
(Fig.
ecules.
Enzymatic
turnovers
of
a
few
motor
E FADH2 + O2
E FADH2 • O2 !oxidized
E activation,
FAD
+1A)
H2was
O
2
he measurerates
under
similar
conditions
(26,
27).
such
an
event
is
usually
much
longer.
The
0
taken
with
an
inverted
fluorescence
microprotein
systems
have
been
monitored
in
real
1') for each
k 1
he
SPU stan- The
scope by raster-scanning
the sample
with a to
time
(19 –21).
In the
study reported
here, by
we
length
of the
trajectories
is limited
emission
on-time and off-time
correspond
0.2
SMOW values
fixed
He-Cd
focus and
enzymatic
fla- kthe
pexamined
[exp(turnovers
k1 t)of single
exp(
t)]“waiting
mit klaser
=fornm,
0theLiCONiX)
time”
FAD reduction
on (t) =
2
1(442
The !17 or
voenzyme molecules by monitoring the fluollows:
oxidation reactions, respectively. The most
„on“-time
(
of 17 or 18O/
to unknown
her details of
will be given
. After SIMS
as evaluated
n microscopy
cron phases
eration prodraters of the
rescence from their active sites. Statistical
analyses of chemical dynamics at the singlemolecule level revealed insights into enzy-
H. P. Lu and X. S. Xie, Pacific Northwest National
Laboratory, William R. Wiley Environmental Molecular Sciences Laboratory, Richland, WA 99352, USA. L.
Xun, Washington State University, Department of
Microbiology, Pullman, WA 99164, USA.
*To whom correspondence should be addressed. Email: [email protected]
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme, 5.1.1 Aktivität
Scheme 1.
tion intensity; thus,
trajectory in Fig. 1B is its stochastic nature.
y to the enzymatic
On a single-molecule basis, the event of a
lectronic state rather
chemical reaction takes place on the subpicoomena. (iii) The avsecond time scale and cannot be time rethe trajectories are
solved here. However, the time needed for
special
substrate,
k2 becomes
e-averagedWith
turnover
diffusion,
thermal activation,
or bothrate
beforelimiting
tions (26, 27).
such an event is usually much longer. The
of kand
ectories is broad
limited bydistribution
emission on-time
2 off-time correspond to
the “waiting time” for the FAD reduction and
oxidation reactions, respectively. The most
Substrate
on-time distribution derived
COx molecule with 2 mM
(-diol substrate (k2 being
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme, 5.1.1 Aktivität
from www.sciencemag.org on September 7, 2008
•
•
Downloaded from www.scien
Static heterogeneity
• Abweichung vom exponentiellen Verlauf
lized in a
from the
as taken in
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme, 5.1.1 Aktivität
a focused
Downloaded from www.scien
Correlation analysis
if we assume k–1 # 0, it follows
c analysis (31) that the probabiln of on-times is the following:
order of k2, we made k2 rate limiting. This
condition is not quite achievable with a high
concentration of cholesterol, but it is achievable with a high concentration of 5-pregene3'-20(-diol substrate, a derivative of choles-
sure the
ensemb
(such a
The flu
the rev
follows
conditional
distribution
on-times (x
ed by a cerf turnovers.
and y axes
1 s. (A) The
l histogram y
of two adjas, which is
he trajectox molecules
5-pregeneubstrate. A
x
al feature is
Consecutive
he 2D conditional histogram
for two on-times separated „On-times“with
by 10 turnovers for the COx
A). The diagonal feature vanishes because the two on-times become independent of
„On-times“
10(B)turnovers
the 10-turnover separation.
The color code in (A) and
represents thespacing
occurrence
I SM Fluo,
Anwendungen, 5.1 Enzyme, 5.1.1 Aktivität
350 Teil(red)
toKap.
0 5(purple).
)c
Dynamic Heterogeneity?
(k 2 $ k 1)
• DoesAactivity fluctuate overBtime?
where )
FAD m
ation ex
able for
equilibr
state of
is in the
averagi
+,(t) #
macrosc
tocorrel
property
from m
!"I(t)"I(0)#, with a decay rate being the sum
of the forward and backward rates. In fact,
fluorescence intensity autocorrelation functions
have
been
used
to
extract
kinetics
„Memory“ in On-times?
Correlation of On-times
•
Teil I SM Fluo, Kap. 5 Anwendungen, 5.1 Enzyme,
Enzyme 5.1.1 Aktivität
2B, das
cay of
arise fr
or both
correlation time of 1.0 , 0.3 s for the fluctuating k2 of this molecule. (B) The r(m) for the
enzyme molecule shown in Fig. 1D with 2 mM
cholesterol. The solid line is a single exponential fitting with a decay constant of 1.2 , 0.5
turnover. With the averaged turnover cycle of
500 ms, we deduce a correlation time of 0.6 ,
0.3 s for the fluctuating k2 of this molecule. (C) The r(m) for the enzyme molecule shown in Fig.
1C with 0.2 mM cholesterol. The averaged turnover cycle of the trajectory is 900 ms. Under this
condition k1 is rate limiting. There is no dynamic disorder in k1.
bution becomes independen
effect arises from a slowly
being rate limiting). The rate
forward reaction in Eq. 1 is
Conformational dynamics???
dP E-FAD/dt % &k 2(
• Spectral fluctuations of FAD on the time scale of
activity fluctuations
Fig. 5. (A) Trajectory of the
spectral mean of a COx molecule in the absence of steroid substrate. Each data
point is the first moment of
the FAD emission spectrum
sequentially recorded with a
100-ms data collection time.
The spectral fluctuation is attributed to a spontaneous
conformational fluctuation.
(B) Emission spectrum of a
COx molecule taken in 100
ms. The spectral mean of the
spectrum is the first data
point in (A). This and other
spectra were taken by using a
combination of a spectrograph (Acton Research, Acton, Massachusetts) and a
cooled intensified chargecoupled device camera with
Gen IV photocathode (Princeton Instruments, Trenton, New Jersey) and corrected for wavelengthTeil I SM Fluo,
Kap. 5 Anwendungen,
Enzyme
Enzyme,
Aktivität function (dots) of the spectral mean for the
dependent
detection
efficiency.5.1(C)
The 5.1.1
correlation
where k2(t) is a stochastic
mean, !k2#. If the fluctuation
k2(t) can be replaced by !k2#
der arises when the time
fluctuation is comparable to
1/k2. The fluctuation of k2(t
terized by the k2 variance
time, that is, the memory tim
We quantitatively analyz
ference between Figs. 3A a
variance parameter (40, 41)
!
n
n
t i t i"m #
i
r(m) !
!
n
n
t 2i #
i
!"t(0)"t(m
!
!"t 2 #
where i is an index number f
m turnovers in a trajectory;
mentally determined on-tim