Intracellular trapping of a cytoplasmic folding intermediate of the phage P22 tailspike using iodoacetamide.
Critical steps in polypeptide chain folding within the bacterial cytoplasm have been difficult to identify. Salmonella cells infected with temperature-sensitive folding mutants of the P22 tailspike protein at restrictive temperature accumulated a metastable folding intermediate with a half-life of 6 min at 39 degrees C. The native trimeric tailspike contains 24 buried cysteines (8/chain) but neither disulfide bonds nor active site cysteines. Eighteen of the 24 cysteines are involved in strong hydrogen bonds (Thomas, G. J., Jr., Becka, R., Sargent, D., Yu, M.-H., and King, J. (1990) Biochemistry 29, 4181-4187). Cyanide and iodoacetamide prevented the folding and association of the restrictive temperature folding intermediate to the native state after shift to permissive temperature. The cytoplasmic folding intermediate was covalently modified by iodoacetamide within infected cells. Chains which had reacted with iodoacetamide were unable to proceed through the folding pathway. Iodoacetamide also reacted with a folding intermediate during the refolding of purified tailspike chains in vitro, inhibiting further folding. No reaction occurred with native tailspike in vivo or in vitro. The target residues in the intermediates were in the carboxyl terminus of the chain and may be a unique set of cysteine residues that are activated during protein folding, but not in the native state.
Sather
S K
SK
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
King
J
J
D017100:Bacteriophage P22 / Q000187:drug effects / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004734:Energy Metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006358:Hot Temperature; D007460:Iodoacetamide / Q000494:pharmacology; D007553:Isotope Labeling; D007700:Kinetics; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000494:pharmacology; D017510:Protein Folding; D012486:Salmonella typhimurium / Q000821:virology; D014764:Viral Proteins / Q000378:metabolism*; D017376:Viral Tail Proteins
D014764:Viral Proteins; D017376:Viral Tail Proteins; D006026:Glycoside Hydrolases; C041312:endorhamnosidase; C083268:tailspike protein, bacteriophage; D011190:Potassium Cyanide; D005033:Ethylmaleimide; D007460:Iodoacetamide