The role of lipids and salts in two-dimensional crystallization of the glycine-betaine transporter BetP from Corynebacterium glutamicum.
The osmoregulated and chill-sensitive glycine-betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for 2D crystallization were compared. Only the use of lipids extracted from C. glutamicum cells led to the formation of large, well-ordered crystalline areas. To understand the lipid-derived influence on crystallinity, lipid extracts from different stages of the crystallization process were analyzed by quantitative multiple-precursor ion scanning mass spectroscopy (MS). Results show that BetP has a preference for fatty acid moieties 16:0-18:1, and that a phosphatidyl glycerol (PG) 16:0-18:1 rich preparation prevents formation of pseudo crystals.
Tsai
Ching-Ju
CJ
Max Planck Institute of Biophysics, Department of Structural Biology, Max-von-Laue Strasse 3, 60438 Frankfurt, Germany.
Ejsing
Christer S
CS
Shevchenko
Andrej
A
Ziegler
Christine
C
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D002308:Cardiolipins / Q000494:pharmacology*; D002352:Carrier Proteins / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology*; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D003460:Crystallization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D007091:Image Processing, Computer-Assisted; D018021:Lithium Chloride / Q000494:pharmacology; D013058:Mass Spectrometry; D008563:Membrane Lipids / Q000494:pharmacology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000187:drug effects; D046529:Microscopy, Electron, Transmission; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000494:pharmacology; D027981:Symporters
D001426:Bacterial Proteins; C101652:BetP protein, Corynebacterium glutamicum; D002308:Cardiolipins; D002352:Carrier Proteins; D002712:Chlorides; D008563:Membrane Lipids; D008565:Membrane Proteins; D027981:Symporters; D012965:Sodium Chloride; D011189:Potassium Chloride; D018021:Lithium Chloride