2d crystallization: reconstitution using hydroxypropyl-β-cyclodextrin ralph lasala, 1 chang kim, 1...
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2D Crystallization: Reconstitution using Hydroxypropyl-β-Cyclodextrin
Ralph Lasala,1 Chang Kim,1 Nicolas Coudray,1 Iban Ubarretxena,2 David Stokes1,3
New York Structural Biology Center1
Mount Sinai School of Medicine2
Skirball Institute, New York University Medical Center3
Introduction:• Successful 2D crystallization screening relies on the general
method of reconstitution of membrane proteins into lipid vesicles.
– removing the detergent from a ternary micellar solution containing purified protein and lipids
• Several parameters affect the formation and the quality of 2D crystals:
– temperature, initial detergent and protein concentration, protein-to-lipid ratio, buffer composition, procedure of detergent removal
• A common method for detergent removal is by dialysis, but it has some limitations:
– longer dialysis time required for low critical micellar concentration (CMC) detergents– in addition, the amount of protein sample often limits the number of crystallization conditions
which can be carried out • Solutions studied to overcome these limitations:
– decrease dialysis time with cyclodextrin• cyclodextrins are cyclic oligosaccharides composed of 6, 7, or 8 glucose units• cyclodextrins have a non-polar cavity which can accommodate hydrophobic or amphiphilic molecules
– reduce the amount of proteins required for crystallization screening using a microdialysis plate
Objectives:- determine the amount of cyclodextrin required for liposome reconstitution
- assess 2D crystallization screening of a membrane protein (Yiip) using cyclodextrin and microdialysis
Example Assay for Reconstitution
vacuum dried lipid film (20 mg DMPC)
add detergent (28 mg DDM)
add dH2O to make 4 ml
aliquot into 8 tubes (0.5 ml each)
each tube:2.5 mg/ml DMPC3.5 mg/ml DDM (2 mM)
add 2-hydroxypropyl-cyclodextrin
add dH2O to make 1 ml per tube
incubate for 1 hour
measure OD500 (Nanodrop)
negative stain EMcarbon-coated gridsglow dischargedstained with 2% uranyl acetate
DMPC
+
detergent
cyclodextrins
cyclodextrin inclusion complexes
Lipid solubilization
Liposome
reconstitution
Analysis
Turbidity Analysis after CD-mediated Reconstitution
Figure 1. Turbidity (optical density, OD, at 500 nm) measurements after addition of cyclodextrin (CD) at increasing molar ratios to DMPC solutions previously solubilized in different detergents (DDM, dodecylmaltoside; OG, octyl-glucoside; DM, decylmaltoside).
Figure 2. Cyclodextrin-mediated reconstitution of DMPC liposomes. Samples were taken for negative staining and examined using JEOL 1230 TEM. Images displayed above show vesicle formation after addition of cyclodextrin at different molar ratios to DDM. Larger versions of some images are shown in the succeeding slides.
EM analysis after CD-mediated Reconstitution
1:0.5
1:1
1:2
DDM:CD
DMPC Reconstitution (1 DDM : 0.5 cyclodextrin)
DMPC Reconstitution (1 DDM : 1 cyclodextrin)
DMPC Reconstitution (1 DDM : 2 cyclodextrin)
Microdialysis combined with Cyclodextrinfor 2D Crystallization of Yiip
Conditions for 2D crystallization of Yiip:0.27 mg/ml DOPG0.54 mg/ml DDM0.45 mg/ml Yiip1:2 DDM: cyclodextrin ratiomicrodialysis at 25oCTES buffer (20 mM TES pH 7.0, 100 mM NaCl, 5 mM MgCl2, 5 mM NaN3)Volume in the dialysis chamber: 10 ul
The Microdialysis Plate (GN Biosystems)
2D crystallization protocol
1) Tubular crystal formation were already observed after a 2-day dialysis with cyclodextrin.
2) Without cyclodextrin, crystals were observed after 3 days
Results
Low amount of proteins required (10 to 30 µl of sample)
Several conditions can be set up in parallel (96 well format)
Suitable for automation (at NYSBC, buffer exchange with a Biomek NXP robot)
Figure 3. Cyclodextrin-mediated 2D crystallization of zinc transporter Yiip (0.27 mg/ml DOPG; 0.54 mg/ml DDM; 0.45 mg/ml Yiip; 1 DDM : 2 cyclodextrin ratio) after 48 hours in microdialysis plate against TES buffer (20 mM TES pH 7.0, 100 mM NaCl, 5 mM MgCl2, 5 mM NaN3) at 25oC.
Yiip Crystallization (using microdialysis and cyclodextrin)
Figure 4. 2D crystallization of zinc transporter Yiip without cyclodextrin (0.27 mg/ml DOPG; 0.54 mg/ml DDM; 0.45 mg/ml Yiip), after 48 hours in microdialysis plate against TES buffer (20 mM TES pH 7.0, 100 mM NaCl, 5 mM MgCl2, 5 mM NaN3) at 25oC.
Yiip Crystallization (using microdialysis only)
Results:• The high turbidity, as measured by OD500 at 1:1, corresponded to the
formation of DMPC lipid vesicles (liposome reconstitution).– at 1:0.5, no fully formed lipid vesicles were observed
• In the 2D crystallization of the zinc transporter Yiip, proteoliposome reconstitution was faster when dialysis was combined with cyclodextrin.
– after 48 hours, 2D crystals in the form of long cylindrical tubes were already formed– without cyclodextrin, no tubes were observed after 48 hours
• Microdialysis reduces the amount of protein sample required for crystallization.
– only 10 µl of ternary (protein+lipid+detergent) solution is required
Conclusions:• Cyclodextrin increases the rate of reconstitution during 2D
crystallization.• Through the microdialysis plate, 2D crystals of Yiip were reconstituted
with only 4.5 µg of proteins.
References:• DeGrip WJ, Vanoostrum J, Bovee-Geurts PH (1998) Selective detergent extraction from mixed detergent/lipid/protein micelles, using
cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes. Biochem J. 330: 667-674.• Signorell GA, Kaufmann TC, Kukulski W, Engel A, Rémigy H-W (2007) Controlled 2D crystallization of membrane proteins using methyl-β-
cyclodextrin. J Struct Biol. 157: 321-328.