Abstract
YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P21, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.
Highlights
Correct membrane insertion, folding and assembly of newly synthesized membrane proteins are essential for their functions
YidC transiently receives the transmembrane (TM) segment released from the Sec translocon (Urbanus et al, 2001; Sachelaru et al, 2013), and prevents protein misfolding induced by nonspecific interactions with other TM segments
A His8 tag followed by a Tobacco etch virus (TEV) protease cleavage site (ENLYFQGQ) was introduced between the 26th and 27th residues of YidC, and the C-terminal 19 and 14 residues followed by the GFP-His8 tag were removed from YidC to produce YidC27–261 and YidC27–266, respectively (Fig. 1a)
Summary
Correct membrane insertion, folding and assembly of newly synthesized membrane proteins are essential for their functions. The functions of YidC in membrane-protein insertion are essential for cell viability (Samuelson et al, 2000). YidC works as a membrane-protein chaperone, which facilitates membrane-protein folding and assembly in cooperation with the Sec translocon. In this Sec-independent pathway, YidC is responsible for the insertion of several single or double membrane-spanning proteins, such as the Fo subunit c of the ATP synthase (Foc), subunit II of cytochrome o oxidase (CyoA) and the mechanosensitive channel MscL. The YidC proteins from Gram-negative bacteria possess an additional TM helix and a large periplasmic region formed by the N-termini of the core TM region. F70, 1056–1060 crystallization communications the large periplasmic domain and electron-microscopic studies (Oliver & Paetzel, 2008; Ravaud et al, 2008; Kohler et al, 2009; Seitl et al, 2014)
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