The extreme hyper-reactivity of selected cysteines drives hierarchical disulfide bond formation in serum albumin.
After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pKaof the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.
Department of Chemical Sciences and Technologies, University of Rome 'Tor Vergata', Italy.
Alessio
A
Bocedi
Department of Chemical Sciences and Technologies, University of Rome 'Tor Vergata', Italy.
Raffaele
R
Fabrini
Department of Biology, University of Rome 'Tor Vergata', Italy.
Jens Z
JZ
Pedersen
Department of Chemical Sciences and Technologies, University of Rome 'Tor Vergata', Italy.
Giorgio
G
Federici
Institute of Biochemistry and Clinical Biochemistry, Catholic University and/or Institute for Molecular Recognition, National Research Council, Rome, Italy.
Federica
F
Iavarone
Institute of Biochemistry and Clinical Biochemistry, Catholic University and/or Institute for Molecular Recognition, National Research Council, Rome, Italy.
Claudia
C
Martelli
Institute of Biochemistry and Clinical Biochemistry, Catholic University and/or Institute for Molecular Recognition, National Research Council, Rome, Italy.
Massimo
M
Castagnola
Department of Chemical Sciences and Technologies, University of Rome 'Tor Vergata', Italy.
Giorgio
G
Ricci
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004285:Dogs; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006041:Goats; D006736:Horses; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D000072417:Protein Domains; D017510:Protein Folding; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D012756:Sheep; D013045:Species Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D005978:Glutathione; D003545:Cysteine
Serum albumin; disulfide; hyper-reactivity of cysteines; oxidative folding; protein structure