Figure 4.
FIG. 4. ITC measurements for lip-LBD binding to wild-type,
H146A- and H291A- human
E1b. ITC experiments were carried out in a MicroCal VP-ITC
microcalorimeter by consecutively injecting aliquots of 1.5 mM
lip-LBD or unlipoylated LBD into the reaction cell containing 25
µM wild-type or mutant human E1b. Binding isotherms for
wild-type ( ), H146A- ' ( o ),
and H291A- ( ) were obtained by
plotting heat changes against the molar ratio of lip-LBD, as
derived from the integrated raw data. The data were fit using
the ORIGIN software supplied by the manufacturer. Wild-type E1b
and the His146- ' variant show similar
affinity for lip-LBD with dissociation constants (K[d]) of 2.52
x 10^-5 M and 1.56 x 10^-5 M, respectively. The binding of the
H291A- mutant to lip-LBD
cannot be detected by ITC as indicated by the absence of heat
changes. Binding of unlipoylated LBD ( ) to wild-type E1b also
cannot be detected.
and H291A- 
human
E1b. ITC experiments were carried out in a MicroCal VP-ITC
microcalorimeter by consecutively injecting aliquots of 1.5 mM
lip-LBD or unlipoylated LBD into the reaction cell containing 25
µM wild-type or mutant human E1b. Binding isotherms for
wild-type ( 
), H146A- 
) were obtained by
plotting heat changes against the molar ratio of lip-LBD, as
derived from the integrated raw data. The data were fit using
the ORIGIN software supplied by the manufacturer. Wild-type E1b
and the His146- 
) to wild-type E1b also
cannot be detected.