Expression, purification and crystallization of a protein resulting from the inversion of the amino‐acid sequence of a helical bundle. Issue 1 (1st January 2017)

• Record Type: Journal Article
• Authors: Kefala, Aikaterini; Kotsifaki, Dina; Providaki, Mary; Amprazi, Maria; Kokkinidis, Michael
• Is Part Of: Acta crystallographica. Volume 73:Issue 1(2017:Jan.)
• Page Start: 51
• Page End: 53
• Publication Date: 2017-01-01
• Subjects: Rop protein -- heptad repeat -- protein folding -- sequence–structure relationship -- hyperthermophilic protein; Crystallography -- Periodicals; Crystals -- Periodicals; 548
• DOI: 10.1107/S2053230X16020173
• Languages: English
• ISSNs: 2053-230X

Abstract

Abstract : To explore the sequence–structure relationships of four‐α‐helical bundles, the revRM6 protein, which has been engineered with the exact inverse amino‐acid sequence of a hyperthermophilic helical bundle, was expressed, purified and crystallized and X‐ray diffraction data were collected. 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 P 41 21 2, with unit‐cell parameters a = b = 44.98, c = 159.74 Å, and diffracted to a resolution of 3.45 Å.