The recombinant human nerve growth factor (hNGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF)
dimers all undergo rapid unfolding and dissociation to
monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase
high-performance liquid chromatography, and size-exclusion
chromatography were used to show that this monomer M1
converts slowly to a more fully unfolded monomer, M2,
by a first order process with half-lives of 22, 2.5, 1.6,
and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively,
at 25 °C. Linear Arrhenius plots for the conversion
of M1 to M2 yielded activation energies
of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and
BDNF, respectively. The refolding of these neurotrophins
from 5 M GdnHCl was also first order with NT-3 the slowest
to refold and BDNF the fastest. Threading of the N-terminus
out through the cystine-knot loop present in each of these
proteins is proposed as the slow step in unfolding. The
number of amino acids in the cystine-knot loop (14 for
hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number
and position of the proline residues in this loop (2 for
hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with
the relative rates of unfolding. The smaller the loop and
the greater the number of prolines, the more hindered and
slower the unfolding.