Reversible disulfide bond formation of intracellular proteins probed by NMR spectroscopy.
Reversible oxidation of amino acids within intracellular proteins leads to local and/or global conformational changes in protein structure. Thus, the enzymatic activity or binding properties of a protein might be regulated by local changes in a cell's redox potential, mediated by the availability of reducing/oxidizing equivalents. Whereas it is well established that intracellular pools of oxidizable groups compensate for oxidative stress, far less is known about the molecular mechanisms that accompany transient and reversible oxidation of cytoplasmic proteins. Therefore, the intrinsic redox properties of proteins amenable to reversible oxidation need to be determined. Here we describe the application of NMR spectroscopy to derive the redox properties of intracellular proteins. As exemplified for thioredoxin 1, the Tnk-1 kinase SH3 domain, and the hSH3(N) domain of the T cell protein ADAP, the conformational changes associated with disulfide bond formation can be followed directly upon titration with different ratios of reduced to oxidized glutathione. Redox potentials can be measured accurately in homogeneous solutions and define the conditions under which regulatory oxidation of the respective protein may occur in the living cell.
Piotukh
Kirill
K
Leibniz Institute for Molecular Pharmacology and Freie Universität Berlin, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Kosslick
Daniela
D
Zimmermann
Jürgen
J
Krause
Eberhard
E
Freund
Christian
C
D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains
D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D013879:Thioredoxins; D005978:Glutathione; D003545:Cysteine