Hydrogen peroxide-induced structural alterations of RNAse A.
Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H(2)O(2) exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H(2)O(2) concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H(2)O(2) concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.
Lasch
P
P
Robert Koch Institute, P 34 Biophysical Structure Analysis, D-13353 Berlin, Nordufer 20, Germany.
Petras
T
T
Ullrich
O
O
Backmann
J
J
Naumann
D
D
Grune
T
T
D006861:Hydrogen Peroxide / Q000494:pharmacology*; D018384:Oxidative Stress; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000187:drug effects*; D017550:Spectroscopy, Fourier Transform Infrared
D006861:Hydrogen Peroxide; D012259:Ribonuclease, Pancreatic