Chemistry Reference
In-Depth Information
Figure 11.1 Three different methods of immobilizing
biomolecules for single-molecule fluorescence studies. Adapted
from [45].
negatively charged in neutral pH, repelling nucleic acids. For studies involving
proteins, BSA surfaces are too adhesive. Therefore, we developed a PEG (polyethyl-
ene glycol)-coated surface that reduces the protein adsorption to an undetectable
level [46
-
48]. Surface passivation using PEG,
first introduced by us for single-
molecule studies [48], has now been used successfully by several other groups as
well as our own [37, 38, 41, 42, 49
56]. If a dense layer of PEG is formed on a quartz
surface (we use amino-silane coating followed by conjugation with PEG modi
-
ed
with NHS-ester at one end), it forms a polymer brush that prevents protein
adsorption to the underlying surface. We incorporate a small fraction of PEG
polymers that are end-modi
ed with biotin to facilitate the immobilization of
biotinylated macromolecules. Proteins interact speci
cally with DNA immobilized
to the PEG surface and their bulk solution activities are well reproduced in all the
systems we have tested [37
-
39, 48, 57]. We have also developed a vesicle encapsula-
tion technique that can measure conformational dynamics of biomolecules free of
surface tethering [58].
11.3
smFRET Studies of Rep Helicase
Helicases are highly ef
cient, processive motor proteins, which are powered by the
hydrolysis of nucleotide co-factors such as ATP. For instance, RecBCD protein can
unwind tens of thousands of base pairs in one run at a speed of
500 bps per
second [59, 60]. Most DNA helicases show directionality in DNA unwinding. Polar
binding to ssDNA and the ability to translocate uni-directionally appear to be its
origin even though the exact relation between ssDNA translocation and unwinding is
not yet clear. Helicases need to couple the conformational changes induced by ATP
binding and hydrolysis to directional motion along a DNA track and unwinding of
