Disulphide-bonded intermediate on the folding and assembly pathway of a non-disulphide bonded protein.
The trimeric parallel beta-coil P22 tailspike contains eight cysteines per chain, but lacks disulphide bonds in the native state, in both the crystalline and solution forms. However, cysteines in a folding intermediate are reactive with thiol blocking reagents, which prevent further productive folding both in vivo and in vitro. The in vivo refolding yield was independent of the availability of metal ions, but was sensitive to redox potential. Isolation by nondenaturing gel electrophoresis of the protrimer intermediate, a trimeric folding intermediate that precedes the fully folded trimer in the in vivo and in vitro pathways, revealed the presence of interchain disulphide bonds. Incubation of the isolated protrimer with reducing agents generated the native trimer. The formation of beta-sheets with interdigitated strands from different subunits in the native trimer may require the transient disulphide bonds for proper alignment. To our knowledge this is the first report of a disulphide bond present in a folding intermediate of a non-disulphide bonded protein.
Robinson
A S
AS
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
King
J
J
D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013462:Sulfur Radioisotopes; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000378:metabolism*
D004220:Disulfides; D008670:Metals; D013438:Sulfhydryl Compounds; D013462:Sulfur Radioisotopes; D017376:Viral Tail Proteins; D004533:Egtazic Acid; D004492:Edetic Acid; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage; D003545:Cysteine