title: Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.
abstract: Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP.
authors.affiliation: Department of Biology, Kenyon College, Gambier, OH 43022, United States. Electronic address: srohrbac@ucsd.edu.
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Rohrback
authors.affiliation: Department of Biological Sciences, Wright State University, Dayton, OH 45435, United States. Electronic address: Michele.Wheatly@mail.wvu.edu.
authors.forename: Michele G
authors.initials: MG
authors.lastname: Wheatly
authors.affiliation: Department of Biology, Kenyon College, Gambier, OH 43022, United States. Electronic address: gillenc@kenyon.edu.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Gillen
mesh_terms: D000818:Animals; D003400:Astacoidea / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing; D012519:Sarcoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D020033:Protein Isoforms; D002118:Calcium
keywords: Affinity; Crayfish; EF hand; Fluorescence; Muscle