Supplemental Research Data

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• Adobe PDF File (167 KB) - H/D exchange experiments
  In order to identify those regions within the pro-peptide that could be involved in contacts with the mature part, mass spectrometry-coupled H/D exchange was employed. This method allows the identification of segments that are protected against H/D exchange due to their engagement in tertiary contacts (Bai et al. 1993; Zhang and Smith 1993). The exchange reaction was performed at 0 °C by dilution of 1 µL of a 250 µM protein solution in 9 µL D2O. After a 2 min incubation interval, the exchange was suppressed by acidification with 90 µL stop buffer (25 mM succinic acid, 25 mM citric acid, pH 2.4). Subsequently, digestion was initiated by adding 1 µL of a 250 µM pepsin solution (Roche Diagnostics). After 45 s, 30 µL methanol was added, and the sample was analyzed by MALDI-ToF. The back exchange of deuterated proNGF during sample preparation was minimized and determined as 12 % by use of control peptides of 12 and 21 amino acids.
  For the alignments of the obtained proteolytic fragments the program FindPept™ (http://www.expasy.ch/tools/findpept.html) was applied.