Refolding of brain-derived neurotrophic factor from guanidine hydrochloride: kinetic trapping in a collapsed form which is incompetent for dimerization.
We have studied the pathway and kinetics of refolding of recombinant human brain-derived neurotrophic factor (BDNF), which is a very tightly-associated dimer in its native state. When BDNF unfolded in 6 M guanidine 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 has extensive secondary structure, but no well-defined tertiary structure. Size-exclusion chromatography with light scattering detection shows that it is compact and monomeric, and therefore corresponds to what is often called a "collapsed form" or "molten globule". This collapsed form disappears with a half-time of approximately 30 min, simultaneously with the appearance of native dimers, without accumulation of monomeric species with a native tertiary structure. Remarkably, the monomer-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 present at the dimer interface of nerve growth factor (and presumably in BDNF), these results indicate that these hydrophobic groups are not exposed in the collapsed form, and that it is therefore quite dissimilar from the native structure. A significant conformational change in the collapsed form is necessary to re-expose these hydrophobic groups to form the dimer interface, making this the rate-limiting step in reaching the native conformation.
Protein Chemistry Department, Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
J S
JS
Philo
R
R
Rosenfeld
T
T
Arakawa
J
J
Wen
L O
LO
Narhi
D019208:Brain-Derived Neurotrophic Factor; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009414:Nerve Growth Factors / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D019208:Brain-Derived Neurotrophic Factor; D006146:Guanidines; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D019791:Guanidine