Biology Reference
In-Depth Information
include mapping of cell surface receptors, probing the mechanical linkage
of membrane receptors to the intracellular structures or extraction of
membrane proteins without using detergents. In this chapter, we describe
methods for mechanically mapping the presence (or absence) of particular
proteins (mainly speciic receptors) on cell membranes using the force
mode of the atomic force microscope (AFM). In such experiments, an AFM
probe is linked to membrane proteins, either with biochemical speciicity
or without, and forces of various magnitudes are applied to the probe
through a force transducer to observe the effect of applied forces to the cell
and cell membrane proteins. Besides AFM, 3,4 force transducers capable of
manipulating membrane proteins at the single-molecule level include laser
tweezers (or optical traps), 5,6 the biomembrane force probe 7 and magnetic
tweezers. 8
The effect of externally applied forces to membrane-bound proteins
has been a subject of intense research in the ield of biomechanics from
the emerging 9 to the present matured phase. 10 Among early attempts to
guide the experimental effort with a sound theoretical base, Bell published
his seminal paper in 1978 11 explaining the on and off kinetics of protein-
protein interactions under the inluence of an applied force. He also gave
an estimate of the force required for “uprooting” the typical intrinsic
membrane protein glycophorin A from lipid bilayers as 250 pN. The idea of
uprooting a irmly anchored membrane protein by force from the biological
membrane was an eye-opening example of the potential of nanotechnology
to many biologists.
Thanks to the invention of laser tweezers and AFM, what was envisaged
by Bell in 1978 has become experimentally veriiable, and many mechanical
experiments on single molecules of DNA, 12-14 RNA, 15 polypeptides, 16,17
synthetic polymers, 18 polysaccharides 19 and proteins 20-27 have been
reported, to name a few examples. In addition to such single-molecule
experiments, measurement of the force required to separate intermolecular
complexes has been done on, for example, complexes between biotin-
avidin, 28 antibody-antigen, 29 lectin-carbohydrate, 30 transferrin-transferrin
receptor, 31 GroEL-unfolded protein 32 and so on.
In force measurements, a key feature is that the tensile strength (the
maximum force to break composite structures by the application of tensile
force) is not a constant for a given sample, but it depends on the loading
rate in a predictable way. 7,33 By taking advantage of the loading rate
dependence of the tensile force, one can determine two parameters related
to the energy diagram of the unbinding reaction, i.e., the dissociation rate
constant under zero external force (natural dissociation rate constant,
k 0 diss ) and the distance for the complex to reach the activated state from its
 
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