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dealt with. Using the external force as an additional knob is very useful in under-
standing the mechanochemistry of motor proteins [92], but the functional para-
meters at the zero force limit can be obtained only through extrapolation because the
resolution deteriorates when the force is reduced.
11.3.3
Different Types of smFRET Approaches to Probe Helicase Mechanisms
11.3.3.1 DNA
DNA FRET
In this study [48], we detected, in real time, DNA binding of a Rep protein and Rep-
catalyzed DNA unwinding by measuring FRET between dyes attached to de ned
DNA locations. The DNA was a duplex with a 3 0 -ssDNA tail denoted as 3 0 -tailed
DNA. The major findings are summarized below and shown diagrammatically in
Figure 11.2.
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(i) In the unwinding experiment, protein and ATP were simultaneously added to
surface-immobilized 3 0 -tailed DNA labeled at the junction with a donor and an
acceptor. FRET, initially near 100%, drops when unwinding initiates. Complete
unwinding is seen as the total disappearance of uorescence because the donor
strand which is not directly tethered to the surface diffuses away quickly. The
unwinding initiation rate re ects the assembly of the functional helicase unit
and exactly matched the rates determined from bulk phase kinetic studies
without dye labels or surface tethering [77].
(ii) In the binding experiment, the two dyes were attached to the extremities of the
ssDNA tail. Remarkably, the binding of a Rep monomer in the presence of ATP
was detected as a burst of FRET fluctuations and the binding saturated at 10 nM
protein concentration. In contrast, the unwinding initiation rate was linearly
dependent on protein concentration above 20 nM, strongly indicating that the
binding of another Rep monomer to a monomer-occupied DNA is the rate-
limiting step in unwinding initiation. That is, a Rep dimer is likely the active
species for unwinding.
Figure 11.2 Initiation and re-initiation of unwinding by Rep. Based
on extensive smFRET data from 3 0 -tailed DNA tethered to a PEG
surface via biotin
streptavidin. Adapted from [48].
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