Domain separation precedes global unfolding of rhodanese.
The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese.
Shibatani
T
T
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760, USA.
Kramer
G
G
Hardesty
B
B
Horowitz
P M
PM
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002417:Cattle; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D013884:Thiosulfate Sulfurtransferase