Abstract
Understanding the nature of protein grammar is critical because amino acid substitutions in some proteins cause misfolding and aggregation of the mutant protein resulting in a disease state. Amino acid substitutions in phage P22 coat protein, known as tsf (temperature-sensitive folding) mutations, cause folding defects that result in aggregation at high temperatures. We have isolated global su (suppressor) amino acid substitutions that alleviate the tsf phenotype in coat protein (Aramli, L. A., and Teschke, C. M. (1999) J. Biol. Chem. 274, 22217-22224). Unexpectedly, we found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates.
Highlights
The process of protein folding is still not completely understood, it is known that larger proteins often have identifiable folding intermediates
We found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates
We have investigated the effect of a global suppressor amino acid substitution on the aggregation propensity of tsf coat proteins in an effort to determine the mechanism of suppression of the tsf folding defects
Summary
Bacteria—The bacteria used for all of the experiments were derivatives of S. typhimurium LT2. Refolding of Coat Protein by Rapid Dilution—Empty procapsid shells were unfolded at 2 mg/ml in 6.75 M urea, 20 mM phosphate buffer at pH 7.6 for 30 min at room temperature. Refolding was initiated by rapid dilution with phosphate buffer to yield a final coat protein concentration of 0.1 mg/ml with 0.34 M residual urea. Refolding of Coat Protein by Dialysis—Empty procapsid shells were unfolded in urea as described above. The samples for the native agarose gel were prepared by combining a portion of the protein with agarose gel sample buffer (40 mM Tris base, 1 mM EDTA, 20% sucrose, pH 8.2, with acetic acid), and ϳ 6 g was loaded onto 1.2% Seakem HGT agarose gel made with the same buffer without the sucrose and run at 50 V for 3.5 h at room temperature [41]. The agarose gels were stained with Coomassie Blue
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