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Abstract
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.
Highlights
Critical stepsin polypeptide chain folding within the in vivo pathway (Seckler et al, 1989;Fuchs et al, 1991; Danner bacterial cytoplasm have been difficult to identifyS. al- and Seckler, 1993)
Iodoacetamide reacted with a folding intermediatteerminal formylmethionine (Sauer et al, 1982).The native tailduring the refolding of purified tailspike chianinvsitro, spike is heat-stable,with a T, of 88 “C
Native tailspike was amide's block of native tailspike formation in vitro was strong not unfolded by either N-ethylmaleimide or iodoacetamide at and reproducible, we focused our attention on the iodoacet- any of the concentrations used
Summary
NATIVE TAILSPIKE have attained enough structure for interchain interactions the same site as 9-amH10142 (Schwarz and Berget, 1989). The cl-7 come together to form the protrimer, a state where the chains are associated but not yet fully folded.This association presumably involves the COOH-terminal domains which are wound around each other in the native structure Media a n d Chemicals-Bacteria and phage were grown and main- Because chain folding both precedes and follows chain association, there is no species corresponding to a "native monomer.". I4C-Labeledamino acid mixture (DuPont-NEN) was ina 2% ethanol solutionwith a specific activity of 54 mCi/milliatom carbon at 100 pCi/ml This mixture was diluted to 2p0CUm1 in M9 salts prior to use. The single chain intermediates, protrimer, and the aggregated state areall sensitive to heat, proteases, and SDS. This last property allows us to follow the formation of native tailexperiments. Further details are given in the text and in the figure legends
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