COMPARATIVE EQUILIBRIUM DENATURATION STUDIES OF THE NEUROTROPHINS - NERVE GROWTH-FACTOR, BRAIN-DERIVED NEUROTROPHIC FACTOR, NEUROTROPHIN-3,AND NEUROTROPHIN-4 5/

The neurotrophins are a family of small dimeric proteins required for the development and survival of vertebrate neurons. Solvent denaturati on 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 i dentity is conserved among this family, significant structural differe nces were revealed by the folding and unfolding of these proteins. Den aturation in guanidine hydrochloride and renaturation at pH 7 and 3.5 were monitored by fluorescence intensity, fluorescence polarization, a nd circular dichroism. The midpoint of equilibrium unfolding curves fo r all four neurotrophins was independent of the technique but was depe ndent on protein concentration, indicating that a two-state model invo lving native neurotrophin dimers and denatured neurotrophin monomers ( N-2 = 2D) describes the equilibrium between folded and unfolded neurot rophins. The conformational stabilities of the dimeric neurotrophins r evealed that mNGF had the lowest conformational stability (19.3 kcal/m ol); hNGF, NT-3, and NT-4/5 had intermediate stabilities, and BDNF had the highest stability (26.4 kcal/mol). Recovery of native spectroscop ic characteristics upon removal of denaturant indicated that the unfol ding process is reversible. Accordingly, unfolding and refolding curve s were coincident for mNGF or NT-4/5 at pH 7 and 3.5 and for BDNF at p H 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 (N-2. = 2I = 2D) be coming populated below pH 4. The differences in stability, pH dependen ce, and coincidence of refolding curves distinguish the homologous str uctures of the neurotrophins.