Nucleic Acids Research, Vol 26, Issue 6 1373-1381, Copyright © 1998 by Oxford University Press
FW Sevenich, J Langowski, V Weiss and K Rippe
Fluorescence anisotropy and fluorescence correlation spectroscopy
measurements of rhodamine-labeled DNA oligonucleotide duplexes have been
used to determine equilibrium binding constants for DNA binding of the
prokaryotic transcription activator protein NtrC. Measurements were made
with wild-type NtrC from Escherichia coli and the constitutively active
mutant NtrCS160Ffrom Salmonella using DNA duplexes with one or two binding
sites. The following results were obtained: (i) the dissociation constant K
d for binding of one NtrC dimer to a single binding site was the same for
the wild-type and mutant proteins within the error of measurement. (ii) The
value of K d decreased from 1.4 +/- 0.7 x 10(-11) M at 15 mM K acetate to
5.8 +/- 2.6 x 10(-9) M at 600 mM K acetate. From the salt dependence of the
dissociation constant we calculated that two ion pairs form upon binding of
one dimeric protein to the DNA. (iii) Binding of two NtrC dimers to the DNA
duplex with two binding sites occured with essentially no cooperativity.
Titration curves of NtrCS160Fbinding to the same duplex demonstrated that
more than two protein dimers of the mutant protein could bind to the DNA.
ARTICLES
DNA binding and oligomerization of NtrC studied by fluorescence anisotropy and fluorescence correlation spectroscopy
Deutsches Krebsforschungszentrum, Abteilung Biophysik der Makromolekule, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
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