Metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF): selectivity and conformational consequences of histidine modification.

We have studied the metal-catalyzed oxidation (MCO) of brain-derived neurotrophic factor (BDNF) with regard to target sites and potential conformational changes of the protein. The exposure of BDNF to three different levels of ascorbate/Cu(II)/O2 [20 microM Cu(II), 2 mM ascorbate (level 1); 20 microM Cu(II), 4 mM ascorbate (level 2); 40 microM Cu(II), 4 mM ascorbate (level 3)], chosen based on the extent of chemical modification of Met and His, respectively, resulted in the exclusive oxidation of a buried Met residue, Met92, at level 1 but in the predominant oxidation of His at level 3. His modification had a significant impact on the structure of BDNF, as quantified by CD and ANSA fluorescence measurements, while Met oxidation had not, also assessed through complementary oxidation of BDNF through hydrogen peroxide. Our ultimate objective was the correlation of the surface exposure of an oxidized His residue in a protein with potential effects on the conformational integrity of the oxidized protein. In a series of three proteins, human growth hormone (hGH), human relaxin (hR1x), and BDNF, we have now observed that His oxidation is paralleled by significant conformational changes when the target His residue is more surface exposed (hR1x, BDNF) while conformational consequences of His modification are less significant when the target His residues are more buried in the interior of the protein (hGH).