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A common mechanism for recombinant human NGF, BDNF, NT‐3, and murine NGF slow unfolding
Author(s) -
De Young Linda R.,
Schmelzer Charles H.,
Burton Louis E.
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.11.2513
Subject(s) - nerve growth factor , recombinant dna , chemistry , neurotrophin , brain derived neurotrophic factor , neurotrophin 3 , biophysics , monomer , neurotrophic factors , endocrinology , medicine , biochemistry , biology , receptor , organic chemistry , gene , polymer
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 sizeexclusion chromatography were used to show that this monomer M 1 converts slowly to a more fully unfolded monomer, M 2 , 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 M 1 to M 2 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.