The neurotrophins are a family of small dimeric proteins required for the development and survival of vertebrate neurons. Solvent denaturation studies were used to compare recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) to nerve growth factor isolated from mouse submaxillary glands (mNGF). Although greater than 50% sequence identity is conserved among this family, significant structural differences were revealed by the folding and unfolding of these proteins. Denaturation in guanidine hydrochloride and renaturation at pH 7 and 3.5 were monitored by fluorescence intensity, fluorescence polarization, and circular dichroism. The midpoint of equilibrium unfolding curves for all four neurotrophins was independent of the technique but was dependent on protein concentration, indicating that a two-state model involving native neurotrophin dimers and denatured neurotrophin monomers (N2 = 2D) describes the equilibrium between folded and unfolded neurotrophins. The conformational stabilities of the dimeric neurotrophins revealed that mNGF had the lowest conformational stability (19.3 kcal/mol); hNGF, NT-3, and NT-4/5 had intermediate stabilities, and BDNF had the highest stability (26.4 kcal/mol). Recovery of native spectroscopic characteristics upon removal of denaturant indicated that the unfolding process is reversible. Accordingly, unfolding and refolding curves were coincident for mNGF or NT-4/5 at pH 7 and 3.5 and for BDNF at pH 3.5. However, BDNF and NT-3 unfolding and refolding curves were not coincident at pH 7. The stability of the neurotrophins decreased as pH decreased, with compact monomeric intermediates (N2 = [2I] = 2D) becoming populated below pH 4. The differences in stability, pH dependence, and coincidence of refolding curves distinguish the homologous structures of the neurotrophins. Study holds ProTherm entries: 4432, 4433, 4434, 4435, 4436, 4437, 4438, 4439 Extra Details: dimeric proteins; two-state model; conformational stability;,refolding curves
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:25 p.m.
|Number of data points||24|
|Assays/Quantities/Protocols||Experimental Assay: Cm buffers:citrate-phosphate: 100 mM, pH:3.5 ; Experimental Assay: m buffers:citrate-phosphate: 100 mM, pH:3.5 ; Experimental Assay: dG_H2O buffers:citrate-phosphate: 100 mM, pH:3.5 ; Experimental Assay: Cm buffers:Na2HPO4,NaH2PO4,: 50 mM,50 mM,, pH:7.0 ; Experimental Assay: m buffers:Na2HPO4,NaH2PO4,: 50 mM,50 mM,, pH:7.0 ; Experimental Assay: dG_H2O buffers:Na2HPO4,NaH2PO4,: 50 mM,50 mM,, pH:7.0|
|Libraries||Mutations for sequence MSILFYVIFLAYLRGIQGNSMDQRSLPEDSLNSLIIKLIQADILKNKLSKQMVDVKENYQSTLPKAEAPREPEQGEATRSEFQPMIATDTELLRQQRRYNSPRVLLSDSTPLEPPPLYLMEDYVGNPVVANRTSPRRKRYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT|