Biology Reference
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$
x
intercept of the plot with the axes,
k
0
off
were determined and the energy
diagram of the reaction was reconstructed. The results obtained on live HeLa
cells were similar to those obtained on the mica surface including the pattern
of loading rate dependency. They also mapped the locations where strong
ligand-receptor interactions were observed with a time lapse of 3.5 minutes
over a 2 μm × 2 μm area, and the result is given in
Fig. 12.3c-e
.
and
12.5 UNBINDING AGAINST UPROOTING OF MEMBRANE
PROTEINS
A key question arises while mapping experiments are being performed,
i.e., “which is a more likely event, unbinding of ligand from its receptor or
uprooting of receptor from the cell membrane?” This is a dificult question
to answer because the force to unbind a ligand-receptor pair and that of
uprooting a membrane protein seems to be in a similar range of several tens
of pNs to 400 pNs.
Experimentally, uprooting, i.e., extracting intrinsic proteins from the cell
membrane, can be done by using bifunctional covalent cross-linkers. Afrin
et al.
modiied an amino-silanized AFM probe with the bifunctional covalent
cross-linkers, disuccinimidyl suberate, and brought the modiied probe in
contact with the surface of living Balb 3T3 ibroblast cells. After allowing the
cross-linkers to react with the amino groups on the cell surface, the cantilever
was pulled up and force curves were recorded for many such trials. The inal
rupture force of the force curves obtained from such trials were collected,
and a mean value was obtained from the Gaussian itting curve to the force
histogram as approximately 450 pN,
53
which was much less than the force to
sever a covalent bond. The mean force value was a little higher than the value
of 250 pN as predicted by Bell for extraction of a dimer of glycophorin A from
the lipid bilayer
11
but still within a fair agreement within a factor of two.
In their later work, Afrin
used a similar method as mentioned
earlier to extract red blood cell membrane proteins after deglycosylation of
the cell surface
54
and obtained a value of 150 pN from the major and ~70
pN from the minor peak as mean values of membrane protein uprooting.
After a brief heating of the cells to denature spectrins, the mean force
became 70-80 pN, indicating that the larger force of 150 pN corresponded
to uprooting of membrane proteins that were initially linked to the spectrin-
based cytoskeletal structure. Since covalent cross-linkers do not react with
selected types of membrane proteins, correspondence of the measured force
to uprooting events of speciic kinds of membrane proteins could not be
established. Because major proteins on the red blood cell surface are limited
et al.
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