The tert-butyl hydroperoxide-induced oxidation of actin Cys-374 is coupled with structural changes in distant regions of the protein.
The susceptibility of monomeric actin to both methionine and cysteine oxidation when treated with the oxidizing agent tert-butyl hydroperoxide (t-BH) was investigated. The results show that no methionine residue was susceptible to oxidation by t-BH at concentrations of 1-20 mM, while Cys-374, one of the five cysteine residues of the actin molecule, was found to be the site of the oxidative modification. Perturbations in the intrinsic tryptophan fluorescence and the decreased susceptibility to limited proteolysis by alpha-chymotrypsin and subtilisin of oxidized actin give an indication of some alterations in protein conformation in subdomain 1, and in the central segment of surface loop 39-51, in subdomain 2. Urea denaturation curves indicate a lower conformational stability for the oxidized actin. G-actin structural alterations due to Cys-374 oxidation produced by t-BH result in a decrease in the maximum rate of polymerization, an increase in both the delay time and the time required for half-maximum assembly, a decrease in the elongation rate, and enhancement of the critical monomer concentration for polymerization. The results suggest that oxidation of actin Cys-374 induces structural alterations in the conformation of at least two different distant regions of the molecule. The involvement of both the C-terminus of the actin polypeptide chain and the DNase-I-binding loop in the intermonomer interactions in the polymer could account for the altered kinetics of polymerization shown by the oxidized actin.
DalleDonne
I
I
Department of Biology, University of Milan, Italy.
Milzani
A
A
Colombo
R
R
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003488:Cyanogen Bromide / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
D000199:Actins; D003432:Cross-Linking Reagents; D016877:Oxidants; D010446:Peptide Fragments; D011108:Polymers; D013438:Sulfhydryl Compounds; D014364:Tryptophan; D014508:Urea; D020122:tert-Butylhydroperoxide; D013381:Subtilisins; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D008239:Lysine; D003545:Cysteine; D003488:Cyanogen Bromide