Chemistry Reference
In-Depth Information
motors. For example, Topoisomerase II removed supercoils that had been created by
twisting DNA through magnetic beads [60]. The removal of the supercoil was
measured by monitoring the expansion between the two DNA ends. Another example
is DNA polymerase, which converts single-stranded DNA into double-stranded DNA.
This DNA conformation change was detected by measuring the extension of DNA at
constant load [61]. The unwinding of DNA and RNA as catalyzed by helicase has also
been measured using mechanical and optical methods (see Chapter 11)
1.8
Imaging with AFM and Force Measurements
AFM [62] is a unique tool for force measurements and imaging, offering single
molecule sensitivity. It has been applied to both single protein [63] and DNA
molecules [64]. In contrast to
fluorescence imaging, which is based on local
information around the
fluorophores, AFM provides topological imaging and
speci
c electrostatic interaction mapping without modifying the biomolecules.
Recently, both temporal and spatial resolutions have been dramatically improved [65]
allowing the dynamic operations of biomolecules and the molecular machines
responsible for their assembly to be visualized in real time (see Chapter 12).
The interaction force between single molecules has been measured for strepta-
vidin-biotin using AFM [66, 67] and the forces involved in ligand
-
receptor com-
plexes, antibody
-
antigen complexes, adhesion of complementary strands of DNA,
carbohydrate
-
carbohydrate complexes, lectin
-
carbohydrate complexes, and cell
adhesion proteins have all been studied (see Chapter 6). AFMand othermanipulation
techniques have also been used to study the folding and unfolding of proteins. In
1997, the unfolding and folding of titin which is an extremely large protein, was
measured when its two ends were pulled and shortened [68
-
70]. Force clamp
methods of AFM allow the detailed processes of individual protein molecule folding
to be scrutinized rather than depending on the stepwise transition observed in
ensemble measurements [71] (see Chapter 13).
References
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2 Orrit, M. and Bernard, J. (1990) Single
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fluorescent molecules and
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fluorescence excitation in a p-terphenyl
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5 Rigler, R. (1995) Fluorescence
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Single molecules observed by near-
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