quiz (2/20/08, chpt 3, ecb)
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Quiz (2/20/08, Chpt 3, ECB)1. A chemical process where there is a net gain of electrons is called _______________. A chemical process where there is a net loss of electrons is called _________________.
2. Enzymes are catalysts, they often _____________ the free energy of a reaction by favoring a transition state.
3. This nucleotide cofactor prominently featured as an electron carriers are _____________ and ______________.
4. This is the constant at which an enzyme is operating at half of its maximum speed. _____________________
5. The maximum number of catalytic cycles an enzyme can perform per unit time is called the _____________.
reduction
oxidation
lower
NAD+ NADP+
Km, Michalis-Menten constant
Turnover rate
Fluorescence Imaging with One Nanometer Accuracy
(1.5 nm, 1-500 msec)
Techniques neededSpecificity to look at heads
Nanometer spatial localization
Second temporal resolution
Single Molecule sensitivity
Single Molecule Photostability
Fluorescence
How to get nanometer localization with visible photons?
in
out
8 nm
Imaging Single Molecules with very good S/N
Total Internal Reflection Microscopy
For water (n=1.33) to air (n=1.0): what is TIR angle?
For glass (n=1.5), water (n=1.33):
what is TIR angle? what is penetration depth? >57°
dp=(/4)[n12sin2i) - n2
2]-1/2
dp = 58 nm
With dp = 58 nm , can excite sample and not much background.
TIR- (> c) Exponential decay
Experimental Set-up for TIR(2 set-ups)
Wide-fieldObjective-TIR
Laser
Objective
Filter
Dichroic
Sample
CCDDetector
Lens
Wide-field, Prism-type, TIR Microscope
Sample
Laser
Objective
Filter
CCDDetector
Lens
In one case, sample is “upside-down.” Does this make a difference? No!
Diffraction limited spotWidth of /2 ≈ 250 nm
center
width
Enough photons (signal to noise)…Center determined to ~ 1.3 nm
Dye last 5-10x longer -- typically ~30 sec- 1 min. (up to 4 min)
Accuracy of Center = width/ S-N = 250 nm / √104 = 2.5 nm= ± 1.25nm
How well can you localize?What does it depend on?
(3 things)
1. # of Photons Detected (N)
3. Noise (Background) of Detector (b)(includes background fluorescence and detector noise)
2. Pixel size of Detector (a)
i
22
2422 812
Na
bs
N
a
N
s ii =
derived by Thompson et al. (Biophys. J.).
center
width
Experimental Setup: Imaging Single MoleculesCy3-DNA Immobilized on coverslip
Cy-3 DyeBiotinylated DNA
Streptavidin
Biotinylated BSA
coverslip
coverslip
DNA Sample
3’
18mer
Move stage
In 8 nm, 16 nm, 37 nm increments
Data: Model System: 30 nm Artificial Steps
0 20 40 60 80 1000
200
400
600
800
Time (sec)
26 28 30 320
5
10
no
. of
step
s
poissonian step size (nm)
26 28 30 320
5
10
no
. of
step
s
regular step size (nm)
= 28.52nm = 1.24nmEx.= 28.55nm
= 29.08nm = 1.32nmEx.= 28.55nm
Po
sitio
n (
nm
)
12 nm steps
0 10 20 30 40 50
0
30
60
90
120
150
10 11 12 13 14 150
1
2
3
4
5
no
. of
step
s
regular step size (nm)
Time (sec)
10 11 12 13 140
1
2
3
no
. of
step
s
poissonian step size (nm)
= 12.47nm = 1.37nm
Ex.= 12.35nm
= 12.26nm= 1.39nmEx.= 12.35nm
Po
sitio
n (
nm
)
8 nm steps
0 10 20 30 40 50
0
20
40
60
80
100
6 7 8 90
1
2
3
4
5
Time (sec)
Po
sitio
n (
nm
)
4 5 6 7 8 90
1
2
3
4
5
no
. of
step
s
regular step size (nm)
= 7.63nm= 0.93nmEx. = 7.45nm
= 6.42nm = 1.09nmEx. = 6.52nm
no
. of
step
s
poissonian step size (nm)
Cys66
Cys73
Center of mass
37/2 nmx
74 nm
37-2x
37 nm
Myosin V Labeling on Light Chain: Expected Step Sizes
Expected step sizeHand-over-hand:
Head = 2 x 37 nm= 74, 0, 74 nmCaM-Dye: 37-2x, 37+2x, …
Inchworm: always Scm = 37 nm
0 nm
37+2x
A Single Myosin V moving
37 nm or 74 nm?
86 nm pixel
[ATP] = 300 nM (Low)
0 10 20 30 40 50 60 70 80 90 1000
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
60 65 70 75 80 85 900
10
20
30
40 = 74.08 nm = 5.25 nm
R2 = 0.99346
2 = 1.6742 N
mol = 31
Nstep
= 227
histogram of 74-0 nm steps
step size (nm)
num
ber o
f ste
ps
75.4 nm
82.4 nm
65.1 nm
83.5 nm
69.8 nm
64.2 nm
78.8 nm
70.0 nm
70.9 nm
79.1 nm
67.4 nm
68.8 nm
73.7 nm
70.7 nm
70.1 nm
68.9 nm
71.3 nm
72.0 nm
time (sec)
Posi
tion
(nm
)
higher [ATP](>400 nM)
[ATP] = 300 nM
Myosin V steps: 74 nm +/- 5nm
+/- 1.3 nm
+/- 1.5 nm
+/- 1.3 nm
52-23 nm steps
0 10 20 30 40 50 60
0
100
200
300
400
500
600
700
800
900 s1= 23.08 nm =3.44 nm N
mol=6
s2= 51.65 nm = 4.11 nm N
step=92
73.4 nm
73.5 nm
69.2 nm
44.8 nm24.9 nm
74.4 nm
72.2 nm
25.6 nm58.1 nm
18.9 nm
45.5 nm
19.2 nm
27.2 nm
74.3 nm
58.6 nm18.7 nm
66.6 nm
82.1 nm
72.5 nm
56.6 nm
68.4 nm
73.5 nm54.1 nm
48.8 nm
15.4 nm48.3 nm
23.7 nm
25.8 nm
68.2 nm
20 30 40 50 60 70 800
2
4n
o.
of
ste
ps
step size (nm)
time (sec)
Pos
ition
(nm
)
[ATP] = 300 nM0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
5
10
15
20
25
30
35
40
duration between adjacent steps (sec)
num
ber
of s
teps
40-30 nm and 50-20 nm dwell time histogram
k = 0.302 R2 = 0.979 Nmol=12 Nstep=191
74-0 nm dwell time histogram
k1 =0.3281 Nmol=37
k2 = 0.3278 Nstep=361
R2 = 0.983
k1=k2 k = 0.3279 ± 0.007
A
74 nm (t sec) observable!observable!
k1 k2
B A
x nm 74-x nm
tketg 2)( tketf 1)(
(t-u) secu sec
A
tkettPkk 21
A A t
duutguftP0
)()()(21
12
kk
ee tktk
74-0 nm Steps: Detecting 0 nm Intermediate by Kinetics
Class evaluation
1. What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
Answer, and turn in at the end of class.
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