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A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding

Published online by Cambridge University Press:  01 November 1999

LINDA R. DE YOUNG ,
CHARLES H. SCHMELZER  and
LOUIS E. BURTON
LINDA R. DE YOUNG
Affiliation:
Department of Pharmaceutical Research and Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 Current address: Angiogenix, Inc., 875-B1 Cowan Road, Burlingame, California 94010.
CHARLES H. SCHMELZER
Affiliation:
Department of Recovery Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080
LOUIS E. BURTON
Affiliation:
Department of Recovery Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 Current address: Bayer Corp., 800 Dwight Way, P.O. Box 1986, Berkeley, CA 94701.
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Abstract

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.

Keywords

cystine-knotguanidine hydrochloridenerve growth factorneurotrophic factorneurotrophinprotein foldingslow unfolding
Type
Research Article
Information
Protein Science , Volume 8 , Issue 11 , November 1999 , pp. 2513 - 2518
Copyright
© 1999 The Protein Society

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