A novel approach for screening the proteome for changes in protein conformation.
Changes in surface hydrophobicity are generally considered as a sensitive indicator for monitoring the structural alterations of proteins that are often associated with changes in function. Currently, no technique has been developed to screen a complex mixture of proteins for changes in the conformation of specific proteins. In this study, we adapted a UV photolabeling approach, using an apolar fluorescent probe, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (BisANS), to monitor changes in surface hydrophobic domains in either purified rhodanese or skeletal muscle cytosolic proteins by urea-induced unfolding or in response to in vitro metal-catalyzed oxidation. Using two-dimensional polyacrylamide gel electrophoresis (2D PAGE), we identified two specific proteins in skeletal muscle cytosol that exhibited a marked loss of incorporation of BisANS after exposure to in vitro oxidative stress: creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the activities of both enzymes were also reduced significantly in response to oxidative stress. We then determined if this method could detect changes in surface hydrophobicity in specific proteins arising from oxidative stress generated in vivo by muscle denervation. A loss in surface hydrophobic domains in CK and GAPDH was again observed as measured by the BisANS photoincorporation approach. In addition, the CK and GAPDH activity in denervated muscle was markedly reduced. These data demonstrate for the first time that this assay can screen a complex mixture of proteins for alterations in surface hydrophobic domains of individual proteins.
Pierce
Anson
A
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
deWaal
Eric
E
Van Remmen
Holly
H
VanRemmen
Holly
H
Richardson
Arlan
A
Chaudhuri
Asish
A
D000818:Animals; D003402:Creatine Kinase / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000378:metabolism; D018384:Oxidative Stress; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020543:Proteome; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism; D013997:Time Factors
D009282:Naphthalenesulfonates; D020543:Proteome; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003402:Creatine Kinase; D013884:Thiosulfate Sulfurtransferase