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Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

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Abstract

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.

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Notes

  1. The detection limit for lipid using TLC is ~1 µg (1 µg lipid = 1 × 10−6 g/750 g mol−1 = 1.3 × 10−9 mol = 1.3 nmol lipid) assuming an average lipid molecular weight of 750 g mol−1. The amount of Δ7 DgkA sample used in the analysis was 120 µg (120 µg = 120 × 10−6 g/14,318 g mol−1 = 8.4 × 10-9 mol = 8.4 nmol DgkA). The lipid content of the protein is therefore less than (1.3/8.4) 0.16 mol lipid per mol Δ7 DgkA.

  2. By comparing the staining intensity in the S30 sample lane (Lane 4) with the lipid standards (Lanes 1–3) in Fig. 5, it is possible to estimate that the lipid in the S30 extract in Lane 4 corresponds to ~3 μg total lipid. 2 μL of extracted S30 lipid dissolved in chloroform was spotted in Lane 4. The total volume of the S30 lipid extract was 20 μL representing 30 μg total lipid derived from 0.3 mL S30 solution. This corresponds to 100 μg lipid/mL S30 extract.

  3. Following the procedure outlined in Footnote 2, the membrane lipid content in dry E. coli cells can be estimated. Lane 5 in Fig. 6 corresponds to ~6 μg total lipid from 2 μL lipid extract. The total volume of the lipid extract was 10 mL which corresponds to 30 mg membrane lipid derived from 1 g wet cells (0.31 g dry cell mass [30]. Thus, the membrane lipid content of dry E. coli cells is estimated at 10 %. A value of 9.1 % lipid has been reported [31].

  4. The approximate cost for a 1-mL cell free reaction is as follows: S30 extract: €0.43, plasmid: €2.14, T7 polymerase: €0.34, amino acids: €0.35, folinic acid: €0.07, RNA inhibitor: €12.43, tRNA: €6.46, pyruvate kinase: €0.28, AcP: €18.33, PEP: €18.40, NTP mix: €8.81, protease inhibitor: €3.33, HEPES: €0.22, PEG 8,000: €0.02, DTT: € 0.02, magnesium acetate, potassium acetate and sodium azide: € 0.04, dialysis cassette: €9.87. Total of €81.54.

Abbreviations

ADP:

Adenosine diphosphate

ARII:

Acetabularia acetabulum rhodopsin II

ATP:

Adenosine triphosphate

C12E8 :

n-octaethylene glycol monododecyl ether

C8E4 :

n-octyl tetraethylene glycol monoether

CL:

Cardiolipin

CTP:

Cytidine triphosphate

CV:

Column volume

DgkA:

Diacylglycerol kinase

DDM:

n-dodecyl-β-d-maltopyranoside

DM:

n-decyl-β-d-maltopyranoside

DNA:

Deoxyribonucleic acid

DTT:

DL-dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

EGTA:

Ethyleneglycoltetraacetic acid

GFP:

Green fluorescent protein

GTP:

Guanosine triphosphate

HCl:

Hydrochloric acid

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HRP:

Horseradish peroxidase

hVDAC1:

Human voltage-dependent anion channel 1

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

kDa:

Kilodalton

LCP:

Lipid cubic phase

LDAO:

Lauryldimethylamine N-oxide

MAG:

Monoacylglycerol

MPD:

2-methyl-2,4-pentanediol

MWCO:

Molecular weight cut off

NADH :

Nicotinamide adenine dinucleotide

OD:

Optical density

PCR:

Polymerase chain reaction

PE:

Phosphatidylethanolamine

PEG:

Polyethyleneglycol

PEP:

Phosphenolpyruvic acid

PK:

Pyruvate kinase

PG:

Phosphatidylglycerol

PIPES :

Piperazine-1,4-bis(2-ethanesulfonic acid

PMSF:

Phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride

RMSD:

Root-mean-square deviation

RNA:

Ribonucleic acid

SEC:

Size-exclusion chromatography

TCEP:

tris (2-carboxyethyl) phosphine hydrochloride

TLC:

Thin-layer chromatography

tRNA:

Transfer ribonucleic acid

UTP:

Uridine triphosphate

YPTG:

Yeast phosphate tryptone glucose

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Acknowledgments

The authors thank J. Lyons, and L. Vogeley for help with diffraction data collection and analysis. This work was supported by grants from Science Foundation Ireland (07/IN.1/B1836, 12/IA/1255), FP7 COST Action CM0902 and the National Institutes of Health (P50GM073210, U54GM094599,). SH and FB were supported by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG) and by the European Instruct consortium. X-ray diffraction data were collected on the 23-ID-B beamline of the General Medicine and Cancer Institute’s Collaborative Access Team (GM/CA-CAT) at the Advanced Photon Source (APS), Argonne, Illinois, USA, the I24 beamline at the Diamond Light Source (DLS), Didcot, Oxford, UK, and PX II at the Swiss Light Source, Villigen, Switzerland.

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Authors and Affiliations

  1. Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

    Coilín Boland, Dianfan Li, Syed Tasadaque Ali Shah & Martin Caffrey

  2. Centre of Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Johann Wolfgang Goethe-University of Frankfurt, Frankfurt, Germany

    Stefan Haberstock, Volker Dötsch & Frank Bernhard

Authors
  1. Coilín Boland
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  2. Dianfan Li
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  3. Syed Tasadaque Ali Shah
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  4. Stefan Haberstock
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  5. Volker Dötsch
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  6. Frank Bernhard
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  7. Martin Caffrey
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Corresponding author

Correspondence to Martin Caffrey.

Additional information

C. Boland and D. Li contributed equally to the work.

Atomic coordinates and structure factors for cell-free-expressed Δ7 DgkA are deposited in the Protein Data Bank under accession code 4D2E.

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Boland, C., Li, D., Shah, S.T.A. et al. Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination. Cell. Mol. Life Sci. 71, 4895–4910 (2014). https://doi.org/10.1007/s00018-014-1655-7

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