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Calorimetic Study of Calcium Binding Proteins with their Interacting Peptides
Author(s) -
Wei ChinChuan,
Schaffer Kyle,
Jensen Drake,
Ngo Rebecca
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.600.8
Subject(s) - isothermal titration calorimetry , chemistry , calmodulin , conformational change , biophysics , peptide , troponin c , binding site , plasma protein binding , ef hand , calcium binding protein , binding domain , biochemistry , crystallography , stereochemistry , calcium , biology , enzyme , psychology , troponin , organic chemistry , psychiatry , myocardial infarction
Several Ca 2+ ‐binding proteins, including Calmodulin (CaM), NADPH Oxidase 5 (NOX5), and dual oxidase (Duox), were employed in calorimetry to understand their structural change upon ion and peptide ligand binding. A general mechanism for these proteins is that Ca 2+ binding triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. Previously, we have characterized chimera proteins constructed by exchanging EF‐hands of CaM with those of Troponin C (TnC) to be used as an informative probe to better understand the functionality of each EF‐hand. Our results revealed that Ca 2+ binding to chimeras impairs their hydrophobicity, and such conformational changes altered their interaction with a CaM‐binding domain (CMBD) peptide of Orai. 3TnC and 4TnC (3 rd and 4 th EF‐hand exchange position, respectively) exhibited a lower ANS binding and solvent accessible surface area change (ΔASA) based on structural homology modeling, but binding to Orai‐CMBD remained similar to CaM, suggesting that the exchanged EF‐hands in the C‐terminal lobe are capable of dynamically accommodating peptide binding. Our isothermal titration calorimetry (ITC) data indicates that approximately 0.3–0.7 protons are released to the buffer when CaM and chimeras are titrated with saturating Ca 2+ , suggesting that key residue(s) involved in Ca 2+ binding are not altered significantly by the exchanged EF‐hands. The heat capacity change of Ca 2+ binding (ΔC p ) for CaM was determined to be ~ −0.66 kCal K −1 mol −1 . The observed large negative value is probably due to 1) the increase in nonpolar ASA and decrease in polar ASA, which results in a positive ΔC p and a higher binding entropy change (ΔS b ) and 2) the reorganization of hydrogen bonds including water molecules (i.e. solvation), contributing a relatively larger negative ΔC p . Interestingly, the ΔC p values of 1TnC and 2TnC are similar to CaM. On the other hand, 3TnC and 4TnC have very small ΔC p values ranging +0.004 to −0.011 kcal K −1 mol −1 , possibly due the decrease of helical content determined by Circular Dichroism. The EF‐hand domain (EFD) of NOX5 has been suggested to be structurally similar to CaM. However, the proton release and ΔC P are ~0.07 and −0.0026 kCal mol −1 K −1 , respectively, indicating a fundamental structural change in CaM that is supported by modeling. We are currently investigating the heat capacity change upon peptide binding for these three Ca 2+ ‐binding proteins, as well as their chimeras and mutants, to give insight about the binding modes and to rationalize apolar/polar surface changes to their ΔC p values. Differential scanning calorimetry (DSC) was also used to gather information about non‐covalent interactions in protein folding. Support or Funding Information This work is supported in part by National Science Foundation CCLI grant and Cottrell College Science Awards (CCSA) from Research Corporation.