Chemistry Reference
In-Depth Information
neck domain is associated with the step movement, in agreement with a walking
model of myosin V [31] (see Chapter 3).
By monitoring
fluorescence changes associated with enzymatic reactions, the
operation of enzymes can be studied. Taking advantage of the cycle between the active
fluorescent oxidized form (FAD) and non-
fluorescent reduced form (FADH) of
avin
adenine dinucleotide, the turnover of cholesterol oxidase was monitored [32]. The
reaction was stochastic, consistent with Michaelis
-
Menten mechanics while a
memory effect was observed between several turnover intervals as a result of slow
conformational isomerizations. Similar memory effects were observed for RNA
enzymes (ribozymes) [27].
Proteins, DNA and RNA ful
ll their function by assembling and disassembling
dynamically. Single molecule spectroscopy is capable of determining the dynamic
structure of particular molecules and molecular interactions in such biomolecular
complexes. A more physiological environment has been observed during protein
folding inmore complex systems such as Gro EL and ES complexes [33]. The dynamic
structures of certain molecular complexes such as helicase, DNA and holiday
junctions, which appear in gene recombination and repair, have been extensively
studied (see Chapter 11).
1.5
Single Molecule Manipulation and Molecular Motors
Laser and magnetic trapping and glass microneedles are common tools used to
manipulate single biomolecules. After Ashkin applied optical trapping techniques
to trap viruses [34], single kinesin was trapped using an attached dielectric bead to
monitor its movement along microtubules immobilized on a glass surface [35],
followed by the
finding of an 8-nm unit step associated with the hydrolysis of single
ATP molecules [36]. Microneedles were
first used to manipulate actin
filaments in
1988 [37]. This led to the measurement of the mechanical properties of actin
filaments and the force generated by both myosin
filaments and single muscle
myosin heads [38]. Actin
filaments have also been manipulated using the laser trap.
Actin
filaments, in which each end was attached to a trapped bead, were manipulated
to interact with single non-processivemusclemyosinmolecules. The stepmovement
of muscle myosin was measured by monitoring the displacement of the actin
filaments [39, 40]. Alternatively, instead of manipulating actin
filaments, single
myosinmolecules have been captured on the tip of amicroneedle andmanipulated to
interact with actin
laments [41]. Themovement ofmyosinwasmonitored directly by
measuring the displacement of myosin. Given that these measurements have
sensitivity to detect thermal motion, the effect of thermal motion on the mechanism
of molecular motor step movement is discussed in Chapter 2.
Unlikemusclemyosin, it is relatively easy tomeasure stepmovement in processive
motors. Manipulation by laser trap has been used for mechanical studies on several
processivemyosinmotors includingmyosinV [42],myosinVI [43] and dynein [44, 45]
demonstrating that ATP hydrolysis reactions are strain-dependent, suggesting that
