Crystallization and preliminary X-ray diffraction analysis of YidC, a membrane-protein chaperone and insertase fromBacillus halodurans

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 sizeexclusion 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 P2 1 , with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å , = 100.3. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

“…Upon IPTG induction, the YidC expression level was greatly improved (SI Appendix, Fig. S10A, LYS, Left) compared with published studies (6) (Fig. 6A, MF lane).…”

“…The MF was then loaded onto the Im7 column in a high concentration of NaCl (0.9 M) and detergent (1.5% dodecyl-maltopyranoside; DDM). A high-purity YidC preparation (∼14 mg protein from ∼20 g cells) was obtained in one-step elution with PSC, much higher than the yield in the published work (6). A small (∼5-7%) but visible impurity was observed in the purified sample (EL; SI Appendix, Fig.…”

“…The structure of YidC has already been determined (6,7). It was selected as a reference in our purification.…”

“…However, our results and those of other investigators show that this approach possesses several drawbacks. It has relatively high, nonspecific, but cooperative affinity to DNA and to DNA-binding proteins (5) as well as to some cellular proteins (4), including many membrane (6,7) and eukaryotic proteins (8).…”

Purification Propensity for Proteins from Bacillus halodurans

“…However, the sensitivity and the specificity behaved oppositely, i.e., the better the sensitivity was, the worse the specificity considerable effort was made to crystallize proteins from B. halodurans [20][21][22]. For instance, E. coli has only one copy of YidC [23] while B. halodurans has two copies of YidC [24], which makes B. halodurans have more benefit for bioengineering. Nevertheless, we hope to focus our attention on the proteins that are more relevant to microbiological applications after this first stage of study.…”