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Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

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 (lambda(ex)/lambda(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 (lambda(ex)/lambda(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.

SUBMITTER: VanScyoc WS

PROVIDER: S-EPMC1302361 | biostudies-other | 2002 Nov

REPOSITORIES: biostudies-other

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Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

VanScyoc Wendy S WS Sorensen Brenda R BR Rusinova Elena E Laws William R WR Ross J B Alexander JB Shea Madeline A MA

Biophysical journal 20021101 5

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 (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is mo ...[more]

PMID: 12414709

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Project description:Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter. Keywords: strain_or_line_design

Dataset Information

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

Publications

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

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