Figure 1.
Fig. 1. Binding of FliM peptide to CheY mutants. A and B,
titration curves of wild-type CheY ( , circle ),
CheY95IA ( , ),
CheY95IM ( , ), and
CheY95IV ( , ) in the
presence and absence of PAM ( filled and open symbols,
respectively). The relative decrease in fluorescence intensity
upon sequential addition of FliM peptide is shown. The data were
fit to a hyperbolic binding function. Note the different scales
on the abscissas in A and B. The inset in A shows Eadie-Hofstee
plots, which were used to determine dissociation constants
(K[D]). The slope of a linear fit to the data yielded K[D]. C,
calculated binding affinities. The values shown in the bar graph
represent the reciprocal of the respective K[D] values
normalized to the K[D] of wild-type CheY in the absence of
phosphodonor. White bars denote the absence, and black bars
denote the presence of PAM. Binding reactions for CheY13DK and
CheY13DK106YW were carried out in the absence of PAM but
displayed in black bars because both proteins presumably
represent the activated conformation (37, 38).
The above figure is reprinted
by permission from the ASBMB:
J Biol Chem
(2000,
275,
19752-19758)
copyright 2000.
, 
circle ),
CheY95IA ( 
, 
),
CheY95IM ( 
, 
), and
CheY95IV ( 
, 
) in the
presence and absence of PAM ( filled and open symbols,
respectively). The relative decrease in fluorescence intensity
upon sequential addition of FliM peptide is shown. The data were
fit to a hyperbolic binding function. Note the different scales
on the abscissas in A and B. The inset in A shows Eadie-Hofstee
plots, which were used to determine dissociation constants
(K[D]). The slope of a linear fit to the data yielded K[D]. C,
calculated binding affinities. The values shown in the bar graph
represent the reciprocal of the respective K[D] values
normalized to the K[D] of wild-type CheY in the absence of
phosphodonor. White bars denote the absence, and black bars
denote the presence of PAM. Binding reactions for CheY13DK and
CheY13DK106YW were carried out in the absence of PAM but
displayed in black bars because both proteins presumably
represent the activated conformation (37, 38).