REFOLDING OF BRAIN-DERIVED NEUROTROPHIC FACTOR FROM GUANIDINE-HYDROCHLORIDE - KINETIC TRAPPING IN A COLLAPSED FORM WHICH IS INCOMPETENT FORDIMERIZATION

We have studied the pathway and kinetics of refolding of recombinant h uman brain-derived neurotrophic factor (BDNF), which is a very tightly -associated dimer in its native state. When BDNF unfolded in 6 M guani dine hydrochloride is diluted 20-fold into phosphate-buffered saline, a partially folded intermediate is rapidly formed (< 1 min). Circular dichroism and fluorescence spectroscopy show that this intermediate ha s extensive secondary structure, but no well-defined tertiary structur e. Size-exclusion chromatography with light scattering detection shows that it is compact and monomeric, and therefore corresponds to what i s often called a ''collapsed form'' or ''molten globule''. This collap sed form disappears with a half-time of approximately 30 min, simultan eously with the appearance of native dimers, without accumulation of m onomeric species with a native tertiary structure. Remarkably, the mon omer-dimer association constant of the collapsed form is approximately 10(10) weaker than the native structure, and it has a low tendency to form large aggregates. Given the very large hydrophobic surface prese nt at the dimer interface of nerve growth factor (and presumably in BD NF), these results indicate that these hydrophobic groups are not expo sed in the collapsed form, and that it is therefore quite dissimilar f rom the native structure. A significant conformational change in the c ollapsed form is necessary to re-expose these hydrophobic groups to fo rm the dimer interface, making this the rate-limiting step in reaching the native conformation.