S-nitrosation of Ca(2+)-loaded and Ca(2+)-free recombinant calbindin D(28K) from human brain.
Calbindin D(28K) is noted for its abundance and specific distribution in mammalian brain and sensory neurons. It can bind three to five Ca(2+) ions and may act as a Ca(2+) buffer to maintain intracellular Ca(2+) homeostasis, but its exact role is still unknown. In the present study, mass spectrometric analysis reveals that the five cysteine residues in recombinant human brain calbindin D(28K) (rHCaBP) are derivatized with N-ethylmaleimide, consistent with the determination of 5.3 +/- 0.4 and 4.7 +/- 0.4 free thiols in the protein using the thiol-specific reagents 5,5'-dithiobis(2-nitrobenzoic acid) and 5-(octyldithio)-2-nitrobenzoic acid, respectively. The results of UV-vis and circular dichroism absorption, intrinsic fluorescence, and mass spectrometry measurements indicate that both Ca(2+)-loaded (holo) and Ca(2+)-free (apo) rHCaBP are S-nitrosated by S-nitrosocysteine (CysNO). The number of cysteine residues S-nitrosated in holorHCaBP and aporHCaBP are 2.6 +/- 0.05 and 3.4 +/- 0.09, respectively, as determined by the Saville assay. HolorHCaBP also undergoes S-nitrosation at one to three cysteine residues when exposed to S-nitrosoglutathione (GSNO), and Cys100 was found to be an S-nitrosation site by peptide mass mapping. Treatment of holorHCaBP with free NO resulted in a mass increase of 59 +/- 2 Da, corresponding to two NO adducts. Since up to four cysteine residues can be S-nitrosated in rHCaBP, it is proposed that the protein may act as a NO buffer or reservoir in the brain in a manner similar to serum albumin in blood. It is significant in this context that rHCaBP is found coexistent with nitric oxide synthase in cerebellum and that S-nitrosation varies with Ca(2+) binding, with S-nitrosation occurring to a greater extent in aporHCaBP than in the holoprotein. Furthermore, exposure of rHCaBP to either CysNO or GSNO also leads to rapid S-thiolation of Cys187. We demonstrate here for the first time that intrinsic protein fluorescence is a sensitive probe of protein S-nitrosation. This is due to efficient Förster energy transfer (R(0) approximately 17 A) between tryptophan donors and S-nitrosothiol acceptors.
Tao
Limei
L
Department of Chemistry and Biochemistry, Concordia University, 1455 de Masisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.
Murphy
Michael E P
ME
English
Ann M
AM
D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D005033:Ethylmaleimide; D006801:Humans; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D026422:S-Nitrosoglutathione; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013439:Sulfhydryl Reagents
D064026:Calbindins; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D013439:Sulfhydryl Reagents; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D003545:Cysteine; D005033:Ethylmaleimide; D002118:Calcium