Cellular and Molecular Life Sciences

, Volume 71, Issue 24, pp 4895–4910

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

  • Coilín Boland
  • Dianfan Li
  • Syed Tasadaque Ali Shah
  • Stefan Haberstock
  • Volker Dötsch
  • Frank Bernhard
  • Martin Caffrey
Research Article

DOI: 10.1007/s00018-014-1655-7

Cite this article as:
Boland, C., Li, D., Shah, S.T.A. et al. Cell. Mol. Life Sci. (2014) 71: 4895. doi:10.1007/s00018-014-1655-7

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.

Keywords

Cell-free Crystallisation In meso Lipid cubic phase Macromolecular crystallography Membrane protein structure 

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

Copyright information

© Springer Basel 2014

Authors and Affiliations

  • Coilín Boland
    • 1
  • Dianfan Li
    • 1
  • Syed Tasadaque Ali Shah
    • 1
  • Stefan Haberstock
    • 2
  • Volker Dötsch
    • 2
  • Frank Bernhard
    • 2
  • Martin Caffrey
    • 1
  1. 1.Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and ImmunologyTrinity College DublinDublinIreland
  2. 2.Centre of Biomolecular Magnetic Resonance, Institute of Biophysical ChemistryJohann Wolfgang Goethe-University of FrankfurtFrankfurtGermany

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