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Biophys J, November 2002, p. 2767-2780, Vol. 83, No. 5
and
*Department of Biochemistry, University of Iowa College of
Medicine, Iowa City, Iowa 52242; Department of Medicine,
Mount Sinai School of Medicine, New York, New York 10029; and
Department of Chemistry, University of Montana,
Missoula, Montana 59812 USA
Cooperative calcium binding to the two homologous domains
of calmodulin (CaM) induces conformational changes that regulate its
association with and activation of numerous cellular target proteins.
Calcium binding to the pair of high-affinity sites (III and IV in the
C-domain) can be monitored by observing calcium-dependent changes in
intrinsic tyrosine fluorescence intensity
(
ex/em of 277/320 nm). However, calcium
binding to the low-affinity sites (I and II in the N-domain) is more
difficult to measure with optical spectroscopy because that domain of
CaM does not contain tryptophan or tyrosine. We recently demonstrated
that calcium-dependent changes in intrinsic phenylalanine fluorescence
(ex/em of 250/280 nm) of an N-domain
fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci.
10:1758-1768). Using steady-state and time-resolved fluorescence
methods, we now show that these excitation and emission wavelength
pairs for phenylalanine and tyrosine fluorescence can be used to
monitor equilibrium calcium titrations of the individual domains in
full-length CaM. Calcium-dependent changes in phenylalanine
fluorescence specifically indicate ion occupancy of sites I and II in
the N-domain because phenylalanine residues in the C-domain are
nonemissive. Tyrosine emission from the C-domain does not interfere
with phenylalanine fluorescence signals from the N-domain. This is the
first demonstration that intrinsic fluorescence may be used to monitor
calcium binding to each domain of CaM. In this way, we also evaluated
how mutations of two residues (Arg74 and Arg90) located between sites
II and III can alter the calcium-binding properties of each of the
domains. The mutation R74A caused an increase in the calcium affinity
of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G
caused an increase in calcium affinity of sites in both domains. This
approach holds promise for exploring the linked energetics of calcium
binding and target recognition.
Biophys J, November 2002, p. 2767-2780, Vol. 83, No. 5
© 2002 by the Biophysical Society 0006-3495/02/11/2767/14 $2.00
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