Journal article
Calcium Binding to Calmodulin Mutants Monitored by Domain-Specific Intrinsic Phenylalanine and Tyrosine Fluorescence
Biophysical journal, Vol.83(5), pp.2767-2780
2002
DOI: 10.1016/S0006-3495(02)75286-7
PMCID: PMC1302361
PMID: 12414709
Abstract
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 (
λ
ex/
λ
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 (
λ
ex/
λ
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.
Details
- Title: Subtitle
- Calcium Binding to Calmodulin Mutants Monitored by Domain-Specific Intrinsic Phenylalanine and Tyrosine Fluorescence
- Creators
- Wendy S VanScyoc - Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242Brenda R Sorensen - Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242Elena Rusinova - Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029William R Laws - Department of Chemistry, University of Montana, Missoula, Montana 59812 USAJ.B. Alexander Ross - Department of Chemistry, University of Montana, Missoula, Montana 59812 USAMadeline A Shea - Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242
- Resource Type
- Journal article
- Publication Details
- Biophysical journal, Vol.83(5), pp.2767-2780
- Publisher
- Elsevier Inc
- DOI
- 10.1016/S0006-3495(02)75286-7
- PMID
- 12414709
- PMCID
- PMC1302361
- ISSN
- 0006-3495
- eISSN
- 1542-0086
- Language
- English
- Date published
- 2002
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Biochemistry and Molecular Biology
- Record Identifier
- 9984024412202771
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