title: The pro-peptide of proNGF: structure formation and intramolecular association with NGF.
abstract: The pro-peptide of human nerve growth factor (NGF) functions as an intramolecular chaperone during oxidative renaturation of proNGF in vitro and interacts intramolecularly with the mature part of native proNGF. Here, we analyzed the structure formation and stability of the pro-peptide in the context of proNGF and its intramolecular interaction with the native mature part. Folding and unfolding of the NGF-coupled pro-peptide, as analyzed by fluorescence, were biphasic reactions with both phases depending on the interaction with the mature part. This interaction was characterized by an overall stability of DeltaG = 20.9 kJ/mol that was subdivided into two reactions, native <--> intermediate state (14.8 kJ/mol) and intermediate <--> unfolded state (6.1 kJ/mol). An additional very fast unfolding reaction was observed using circular dichroism (CD), indicating the presence of at least two kinetically populated intermediates in the unfolding of proNGF. The part of the pro-peptide involved in the intramolecular association with mature NGF comprised the peptide Trp(-83)-Ala(-63) as determined by H/D exchange experiments. Spectroscopic analyses revealed that on the NGF side, a surface area around Trp(21) interacted with the pro-peptide. Trp(21) also participates in binding to TrkA and p75 receptors. These overlapping binding sites of the pro-peptide and the NGF receptors might explain the previously observed lower affinity of proNGF to its receptors as compared to NGF.
authors.affiliation: Martin-Luther-Universität Halle-Wittenberg, Institut für Biotechnologie, 06120 Halle, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Kliemannel
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Schwarz
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
mesh_terms: D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D020932:Nerve Growth Factor / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry*; D020800:Receptor, Nerve Growth Factor / Q000737:chemistry; D020917:Receptor, trkA / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D010455:Peptides; D011498:Protein Precursors; D020800:Receptor, Nerve Growth Factor; C061397:pro-nerve growth factor, human; D020932:Nerve Growth Factor; D020917:Receptor, trkA