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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Farheen
Khan (Vince LiCata, LSU Dept. of Biological Sciences)
A Glimpse Into the Denatured States of Klentaq and Klenow
Klentaq and Klenow are the so called “large fragments”
of the Type I DNA Polymerases of Thermus aquaticus (Taq) and
Escherichia coli (Pol 1), respectively. Previous experiments
in this laboratory have shown that the transition region slopes
(m-values) for Klentaq and Klenow in chemical denaturations
differ from one another. In several other protein systems, the
m-values have been proposed to correlate to the amount of exposed
surface area upon unfolding. However, exceptions to this correlation
have also been reported. Since the transition region slope for
Klentaq denaturation is double that for Klenow and the native
states of both proteins are very similar, the correlation predicts
that Klentaq will expose more surface area as it unfolds. We
are using analytical ultracentrifugation to examine the expansion
of Klentaq and Klenow upon chemical denaturation with guanidine
hydrochloride. In analytical ultracentrifugation, macromolecules
are subjected to a centrifugal field in which the sedimentation
is monitored spectrophotometrically. Sedimentation coefficients,
which are indicative of the size and shape of the macromolecule,
are obtained from sedimentation velocity experiments run in
the analytical ultracentrifuge. Preliminary analysis of the
sedimentation measurements for the chemical denaturations of
Klentaq and Klenow suggest that these two polymerases expand
to a similar extent as they unfold.
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