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. 2010 Jun 2;19(8):1525–1533. doi: 10.1002/pro.431

Figure 5.

Figure 5

Analysis of Dra Snf2 intein branched intermediates in vitro. Branched intermediates (MIP*) accumulated in three different Dra Snf2 intein mutants: Part A, the C-terminal Asn343 was mutated to Ala, Part B, the penultimate His342 was mutated to Ala, and Part C, a triple mutant (Cys323Ser/ Asn343Ala/ Thr+1Ala). Samples were purified by affinity chromatography on Amylose resin and adjusted to pH 6, 9, or 10. Samples were then incubated overnight at the indicated temperatures to examine the breakdown of the BI (thio)ester linkage to yield M + IP in response to pH, temperature or DTT. The time zero sample has a “U” above the lane. Samples were electrophoresed in SDS-PAGE and stained with Coomassie blue. The Asn343Ala and triple mutants were incubated in presence (+) or absence (−) of 50 mM DTT; the His342Ala mutant was incubated in presence of 50 mM DTT. Abbreviations as in Figure 4.