Journal article
Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions
Proteins, structure, function, and bioinformatics, Vol.50(3), pp.381-391
02/15/2003
DOI: 10.1002/prot.10281
PMID: 12557181
Abstract
Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9 degrees C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., [Biochemistry 2002;41:15-20]), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5 degrees C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of 15N-PCaM1-74 and 15N-PCaM1-80 and analysis of high-resolution structural models suggest these residues are latched to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains.
Details
- Title: Subtitle
- Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions
- Creators
- Laurel A Faga - Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109, USABrenda R SorensenWendy S VanScyocMadeline A Shea
- Resource Type
- Journal article
- Publication Details
- Proteins, structure, function, and bioinformatics, Vol.50(3), pp.381-391
- Publisher
- United States
- DOI
- 10.1002/prot.10281
- PMID
- 12557181
- ISSN
- 0887-3585
- eISSN
- 1097-0134
- Grant note
- R01 GM 57001 / NIGMS NIH HHS
- Language
- English
- Date published
- 02/15/2003
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Biochemistry and Molecular Biology
- Record Identifier
- 9984024514502771
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