Expression, purification and crystallization of a protein resulting from the inversion of the amino-acid sequence of a helical bundle

螺旋束氨基酸序列反转产生的蛋白质的表达、纯化和结晶

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Abstract

Earlier studies have found that the occurrence of inverse sequence identity in proteins is not indicative of three-dimensional similarity, but rather leads to different folds or unfolded proteins. Short helices, however, frequently keep their conformations when their sequences are inverted. To explore the impact of sequence inversion on long helices, revRM6, with the inverse amino-acid sequence relative to RM6, a highly stable variant of the ColE1 Rop protein, was engineered. RM6 is a highly regular four-α-helical bundle that serves as a model system for protein-folding studies. Here, the crystallization and preliminary crystallographic characterization of revRM6 are reported. The protein was overexpressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 44.98, c = 159.74 Å, and diffracted to a resolution of 3.45 Å.

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