Equilibrium denaturation of dimeric mouse beta-nerve growth factor (beta-NGF) has been studied by monitoring changes in the protein's spectroscopic characteristics. Denaturation of beta-NGF in guanidine hydrochloride and urea resulted in an altered intrinsic fluorescence emission spectrum, fluorescence depolarization, and diminished negative circular dichroism. Native-like spectroscopic properties and specific biological activity are restored when denaturant is diluted from unfolded samples, demonstrating that this process is fully reversible. However, refolding of denatured beta-NGF is dependent on the three disulfide bonds present in the native protein and does not readily occur when the disulfide bonds are reduced. Graphical analysis and nonlinear least-squares fitting of beta-NGF denaturation data demonstrate that denaturation is dependent on the concentration of beta-NGF and is consistent with a two-state model involving native dimer and denatured monomer (N2 = 2D). The conformational stability of mouse beta-NGF calculated according to this model is 19.3 +/- 1.1 kcal/mol in 100 mM sodium phosphate at pH 7. Increasing the hydrogen ion concentration resulted in a 25% decrease in beta-NGF stability at pH 4 relative to pH 7. Study holds ProTherm entries: 10369, 10370, 10371, 10372, 10373, 10374 Extra Details: beta-nerve growth factor; denaturation; fluorescence; protein structure
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:40 p.m.
|Number of data points||11|
|Proteins||Beta-nerve growth factor ; Beta-nerve growth factor|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O pH:4.0, prot_conc:- ; Experimental Assay: m prot_conc:18.8 micro M ; Experimental Assay: dG_H2O prot_conc:18.8 micro M, pH:7.0 ; Experimental Assay: m prot_conc:6.24 micro M ; Experimental Assay: dG_H2O prot_conc:6.24 micro M, pH:7.0 ; Experimental Assay: m prot_conc:3.76 micro M ; Experimental Assay: dG_H2O prot_conc:3.76 micro M, pH:7.0 ; Experimental Assay: m prot_conc:1.50 micro M ; Experimental Assay: dG_H2O prot_conc:1.50 micro M, pH:7.0 ; Experimental Assay: m prot_conc:1.25 micro M ; Experimental Assay: dG_H2O prot_conc:1.25 micro M, pH:7.0|
|Libraries||Mutations for sequence GEFSVCDSVSVWVGDKTTATDIKGKEVTVLAEVNINNSVFRQYFFETKCRASNPVESGCRGIDSKHWNSYCTTTHTFVKALTTDEKQAAWRFIRIDTACVCVLSRKA|