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Multiple Approach to Determine Protein‐Protein Binding Affinity of Calcineurin Homologous Protein Isoforms 1 and 2 and the Sodium Hydrogen Exchanger Isoform 1
Author(s) -
Marshall Chelsea N,
Wallert Mark Anthony,
Provost Joseph John
Publication year - 2017
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.31.1_supplement.760.22
Subject(s) - chemistry , biochemistry , gene isoform , protein–protein interaction , calcium binding protein , sodium–hydrogen antiporter , biophysics , biology , calcium , sodium , organic chemistry , gene
Protein‐protein interactions direct many important cellular functions. The ability to characterize and quantitate these interactions is importance when studying the mechanisms of biological processes. We are interested examining protein protein interactions experiments between Calcineurin Homologous Protein Isoforms 1 and 2 (CHP1&2) with the Sodium Hydrogen Exchanger 1 (NHE1). NHE1 is a ubiquitous transmembrane protein shown to be of importance in lung cancer proliferation and metastasis. CHP1 and CHP2 are calcium binding proteins that interact with proteins on the plasma membrane and nuclear proteins/transcription factors. Both isoforms share a 61% amino acid sequence homology, both bind to nearly the same residues of NHE1 and have been suggested to bind NHE1 with different levels of affinity. Thus to understand the nature of CHP1/2 – NHE1 interactions we wish to develop a quantitative interaction assay. However, CHP proteins possess several solvent exposed hydrophobic residues making expression, stable solubility and precipitation problematic. To investigate the interaction between these proteins, we have generated several fusion proteins: GST‐NHE1‐CBD (CHP binding domain), 6X His CHP1, 6XHis CHP2 and RFP/GFP His tagged CHP1 and CHP2. We investigated protein‐protein interactions using fluorescent thermal shifts, bait‐pray pull down assays, circular dichroism thermal shifts and quartz crystal microbalance measurements to determine the application of each technique to determining binding affinities.