Biophysical properties of the extra-cellular domain of the calcium-sensing receptor.
The Calcium-Sensing Receptor (CaSR) is a G-protein-coupled receptor that regulates calcium homeostasis by altering parathyroid hormone release, and which binds divalent and trivalent cations, amino acids, polyamines, and polycationic ligands. To obtain information about the structural properties of the CaSR, we expressed milligram quantities of a pure, homogeneous, and functional fragment of the human CaSR extracellular domain (residues 20-535). The expressed and purified protein is folded and binds both neomycin and calcium. It forms dimers in the absence of reducing agents such as beta-mercaptoethanol. Thermal denaturation studies show it has enthalpy and entropy values of unfolding equal to DeltaH=-178+/-4 kJ/mol and DeltaS=-535+/-13 J/mol/K. The protein has significant secondary structure with alpha-helical, beta-sheet, beta-turns, and disordered content of 36.6+/-6.7%, 13.3+/-5.3%, 20.2+/-3.3%, and 29.4+/-4.0%, respectively. The described method for the expression and purification of CaSR should prove useful for further structural studies of this physiologically important protein.
Ryan
Zachary C
ZC
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.
Craig
Theodore A
TA
Venyaminov
Sergei Yu
SY
Thompson
James R
JR
Kumar
Rajiv
R
D000818:Animals; D001703:Biophysics / Q000379:methods; D002118:Calcium / Q000737:chemistry; D049109:Cell Proliferation; D002942:Circular Dichroism; D006801:Humans; D007313:Insecta; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
D044169:Receptors, Calcium-Sensing; D043562:Receptors, G-Protein-Coupled; D002118:Calcium