Today’s Topic: AFM
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Experimental Approach via Atomic Force Microscopy
Imaging Mode, Force Mode.
Example: Measuring strength of Heart Muscle (Titin)
Strength of a single Covalent Bond
Imaging: Correlation Functions
AFM — Force
Force – one place
http://cp.literature.agilent.com/litweb/pdf/5990-3293EN.pdf
http://www.home.agilent.com/agilent/editorial.jspx?cc=US&lc=eng&cke
y=1774141&nid=-33986.0.02&id=1774141
Reversible Unfolding by AFM Pulling on Titin
Why does curve look like
it does?
Why non-linear?
Why repeat?
Does repeat tell you
anything about polymer?
Reversible Unfolding of Individual Titin Immunoglobulin
Domains by AFM, Science, M. Reif, H. Gaub, 1997
Gold
Simple model: Upon reaching a
certain force (peaks, e.g. 1), the
abrupt unfolding of a (Titin)
domain lengthens the
polypeptide by 28 to 29 nm and
reduces the force (troughs) to
that of the value predicted by the
force extension curve of the
enlarged polypeptide (2). Start on
next domain. As it’s pulling,
polymer behaves like WLC.
Example of AFM-Force: Muscle & Titin
The Sarcomere: unit of muscle
Myosin head binds to actin;
rotates upon ATP binding,
pulls actin together.
Myosin II moves Actin
Notice: ATP induces a conformational change: rotation of lever arm
Myosin II acting as a fulcrum, rotating with ATP while driving actin
2 heads of Myosin II; only one head per dimer active.
Myosin II spends about 5% of it’s time bound to actin.
Myosin II is a non-processive motor, i.e. by itself, it takes 1 step on actin.
is processive only because it works in groups
which are held together via the thick filament.
(Vale & Milligan, Science)
Titin: Human’s Biggest protein
Each domain IgG
Silicon Nitride lever:
10’s pN – several
nN’s measureable
Titin: 4.2MDa; Gene (on # 2) = 38,138 aa: Goes from Z-disk to Center; stretchy =I
Band
Picking up a single protein
“needle in a haystack”: usually pick up > 1 protein
“Fingerprint” of e.g. (I91)8: by using identical repeats, unfolding
forces are nearly identical with peaks equally spaced. (see Fig d)
Protein stretched
at constant velocity
Titin: ≈ 1 um/sec
Physiological range
Worm-like Chain (WLC) is very good approximation to
F vs. x of individual unit (protein, DNA) expansion.
Position sensitive detector (PSD)
Useful in AFM, Optical Traps…
Out1
P
In1
N
N
P
Out2
SIGNAL
ΔX ~ (In1-In2) / (In1 + In2)
POSITION
Over a fairly wide
range, it’s linear
ΔY ~ (Out1-Out2) /(Out1+Out2)
In2
Force Spectroscopy
How Strong is a Covalent Bond?
Recall: what did we say it was?
About 100-200 kBT
Covalent bond to the tip, substrate-- gold or glass-- and within Amylose.
We stretched the molecules until one of the covalent bonds in series
ruptured. By analyzing the bond rupture, we were able to identify the bond
that failed. Within amylose (covalent bonds) was found not to rupture.
Note: It’s actually the C-Si which breaks!
How Strong is a Covalent Bond?
Gaub, Science, 1999
Figure 2 (A) Force versus extension curve of amylose covalently bound between an AFM tip
and a silicon oxide surface.
Control: no covalent attachment with amylose.
Reversible stretching of amylose (polysaccharides).
Not dependent on rate of stretching. Sugar rings
switch into a more extended arrangement. With
amylose, this results in a characteristic plateau at 275
pN with an extension of 0.5 Å per ring unit (Fig. 2A).
Thus, this transition can be used as a molecular strain
gauge that can be built into an experiment to report the
force that is acting on any point of the molecular bridge.
With amylose: 275 pN (low-force) with an
extension of 0.5 Å per ring unit
= (275pN)(0.05nm) = 13.75 pN-nm = 3kBT
2B: Covalent attachment: sudden ruptures about 2 pN.
At the given force-loading rates of 10 nN/s, the
histogram peaks at a value of 2.0 ± 0.3 nN.
M Grandbois et al. Science 1999;283:1727-1730
Published by AAAS
Figure 3 (A) Histogram of the length gain after the events were measured in the force versus
extension curves showing multiple ruptures for amylose, which was covalently attached to
the silicon oxide surface and the tip.
Pop, pop, pop
M Grandbois et al. Science 1999;283:1727-1730
Published by AAAS
No EDC or
NHS used.
Attachment
non-specific:
lower force.
A Single Covalent bond
F = 2.0 nN = 2000pN
C-Si: 0.185 nm (estimate)
(2000pN)(0.185 nm) =
370 pN-nm
1kBT = 4pN-nm
E = 92.5 kBT
Sulfur-gold anchor ruptured
at 1.4 +/- 0.3 nanonewtons
at force-loading rates of 10
nanonewtons/second.
Example Rupture Force
Breaking of a covalent bond
C-C ≡ 1600 pN
Breaking of a non-covalent bond.
Biotin/streptavidin ≡ 160 pN (strongest known)
Breaking of a weak bond.
Hydrogen bond ≡ 1- 4 pN
Which is Covalent Bond that breaks?
Largely theoretical argument.
Four bonds are unique to the attachment: Si–O, Si–C, C–C, and C–N bonds. The C–O bond is
found in the attachment and in the amylose backbone. At first, it was difficult to decide which of
these four different bonds was breaking in our experiment. We ruled out the rupture of the Si–O
bond because three of these bonds hold in parallel at the surface. As a first approximation, we
correlated the strength of a covalent bond with the ratio of the dissociation energy and the bond
length. Considering the enthalpy for dissociation and the bond length (20), we decided that the
Si–C bond was the most likely candidate for rupture in our experiment.
How Strong is a Covalent Bond?
Gaub, Science, 1999
AFM Images
Bacteria
DNA molecules
Mosquito eye
http://www.afmhelp.com/index.php?option=com_conten
t&view=article&id=51&Itemid=57
Convolution of tip and sample size
Tobacco Mosaic Virus (TMV)
In truth, diameter of 180 Å.
Due to finite tip size, w~ 350 A
http://webserv.jcu.edu/chemistry/faculty/waner/research/AFM/tipconv.htm
If tip size is large,
have to worry about distortions.
Convolution
What will image look like?
Typically, probe radius varies from 5 to 20 nm
http://webserv.jcu.edu/chemistry/faculty/waner/research/AFM/tipconv.htm
Correlation functions
<f(t) * g(t-t)>
Cross-correlation
Can also do auto-correlation:
(as in WLC)
Crosscorrelation
What if red curve is like a delta function (really narrow)?
Reproduce blue box
What does
crosscorrelation
look like?
http://www.scholarpedia.org/article/1/f_noise
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.