Expression, purification and crystallisation of membrane proteins

Bernadette Byrne

Dept. of Biological Sciences, Imperial College London, UK

Membrane protein structures

Approx. 29,000 protein structures in the PDB-about 90 are integral membrane proteins. Difficulty involved in working with membrane proteins. Researchers often reluctant to take on such expensive and high risk projects-becoming less of a problem

Overview of talk
Expression of membrane proteins Solubilisation of membrane proteins Purification of membrane proteins Practical approach to membrane protein crystallisation Sample preparation Detergent selection Rational screening of crystallisation conditions Optimisation Antibody fragments Trapping single conformations

Membrane protein expression

Some membrane proteins express naturally at very high level e.g. respiratory complexes, photosynthetic complexes, rhodopsin Most membrane proteins require recombinant expression systems allows controlled high level expression addition of affinity tags

Membrane protein expression

Problems: loss of affinity tag inclusion bodies cytotoxic effects homologous expression often not possible heterologous expression often not viable

Heterologous expression = 1 structure

Example
11 ABC transporter proteins Used pET vectors-T7 inducible promoters C-term or N-term His tags 1 was cytotoxic 3 would not express with N-term His tag 2 would not express with C-term His tag (cleavage) 1 formed inclusion bodies High-throughput approach 10 SecY complexes Methanococcus jannaschii

MsbA

Solubilisation
Membrane proteins comprised of hydrophobic and hydrophilic regions Crystallisation requires a protein in a soluble state Detergents solubilise membrane proteins

Solubilisation
Detergent monomer Hydrophilic head Hydrophobic tail

Detergent micelle

+
Detergent at CMC

Detergents
Detergents are monomerically distributed until they reach a threshold concentration where they spontaneously form micelles (CMC). In this state detergents are effective at solubilising (conc. = 1-3 x CMC). CMC is inversely related to the size of the alkyl chain. CMC is sensitive to both temperature and salt conc.

CMC of commonly used detergents

Detergent LDAO octylglucoside dodecyl maltoside Triton X-100 CHAPS Thesit CMC (mM) 2.0 19 0.17 0.23 8 0.09

Note: CMC values for other detergents can be found in the Anatrace catalogue.

Choice of detergent
Two major considerations: Recovery and Stability
CH 2 OH O OH HO OH CH 2 OH O OH HO OH O OH OH Dodecyl-B-D-maltoside (C12 detergent) Octyl- -D-glucopyranoside (C8 detergent)

CH 2 OH

Choice of detergent
Recovery -dependent on expression level Some detergents mimic the membrane better Detergent must be present throughout further handling Detergent used for solubilisation not necessarily the same as that used for crystallisation

Purification of membrane proteins

Membrane proteins tend to have interactions with chromatographic mediastronger

Affinity chromatography often the method of choice. Over-purification of large multi-subunit membrane proteins a potential problem-subunits can be lost.

Purification of membrane proteins

Exploit inherent properties of the protein: Nar, Fdh-N, PS2, cytochrome c oxidase, bc1 complex, SQR His tagged: LacY, cytochrome bo3 ubiquinol oxidase Removal of the His tag??

Purity of sample
Aim for 85-95 % purity But. Attempt crystallisation if only lower purity sample available

Formate dehydrogenase-N

Types of membrane protein crystals

Lipid 2D crystal

Solubilisation & purification Detergent Type I 3D crystal

Membrane protein

Detergent

Lipid cubic phase

Type II 3D crystal

Detergents
Detergents have 2 impeding influences on crystal formation: i) limit the formation of crystal contacts to the hydrophilic regions of the protein. ii) the detergent micelle must fit perfectly into the gap formed within the crystal lattice.

Note: Detergents can be useful additives to the crystallisation of soluble proteins

Practical approach to membrane protein crystallisation

Sample preparation-pure homogenous sample rqd. Detergent selection Mono-dispersity Crystallisation Detergent selection Method Precipitants Temperature Screening kits Optimisation

Sample preparation : Protein concentration and detergent exchange

Best to prepare samples in low buffer concentrations without salt. Reasonable concentration = 10 mg/ml (100-200uM). Large protein complexes may need to use up to 50-80 mg/ml.

Monodispersity
Aggregation is a problem for membrane protein crystallisation DLS is not a suitable method for membrane proteins Negative stain EM Ultracentrifugation

Negative stain EM
(a)
0.5 m

(b)

0.5 m

Crystals
Sample spun at high sped to remove aggregates Before After

Crystallisation 1 : Detergent selection

Selection of detergent is the most critical parameter Often membrane proteins are happier in detergents with longer alkyl chains But detergents which form smaller micelles can leave more of the protein exposed. Choice based on previous experiences or screening

Examples of successfully used detergents

Protein Light harvesting complex Cytochrome bc1 complex Ubiquinol oxidase Cytochrome b6f Nar Detergent OG 1% DDM 0.03% OG 1% DDM 0.2 mM OG 1%

Detergent selection

Detergent combinations
TolC crystallised in 0.6% mix: dodecyl glucoside hexyl glucoside heptyl glucoside octyl glucoside

Rational screening of crystallisation conditions

Vapour diffusion using either sitting or hanging drop. Disadv. Expensive in terms of pure protein and plasticware. Batch method under oil also has a good track record with membrane proteins. Adv. Smaller volumes of protein possible and much less plasticware used. Robotics are being used-use tiny volumes (20 nl) and can test many conditions.

pH

Protein concentration

Salt conc.

Precipitants and temperature

PEG is the most successful precipitant in the presence of non-ionic detergents. Ammonium sulphate and potassium phosphate used for proteins in ionic detergents. Membrane proteins tend to be unstable in high concentrations of organic solvents such as MPD. Crystallisation trials should be performed at at least two temperature- rec. 4C and 20C. Another temperature if possible.

Precipitant

Screening Kits
Screening kits available from Hampton Research and Molecular Dimensions: Crystal screen I Crystal screen II Memb Fac Memb Start Memb Sys Detergent screens Additive screens

Optimisation
Probability of obtaining crystals from the first screen is very small. Possible to vary pH, type and conc. of precipitant Most success achieved by varying the detergent or the addition of additives (commercial screens available). Only accurate test of crystal quality is X-ray analysis. Synchrotron radiation must be used.

Increasing area available for crystal contacts

Crystal contacts limited to exposed regions of protein molecules Two methods: 1) Decrease size of detergent micelle 2) Increase size of protein

Altering the micelle size

1) Exchange of detergent eg DDM to OGcytochrome bo3 ubiquinol oxidase 2) Mixture of detergents- TolC 3) Addition of small amphiphiles eg heptane triolreaction center from Pseudomonas viridis, rhodopsin

Increasing the size of the protein molecules

Addition of a structure specific epitope-antibody fragment. Mice immunised against the protein of interest. Spleen cells fused with an immortalised myeloma cell line-HYBRIDOMA Antibodies secreted into the media Screen essential

Antigen binding site VL

VH CH Hinge region
SS

VH CH CL
SS

Antigen binding site VL

Antibodies
Light chain

CL

SS SS SS SS

Enzymatic cleavage
VH

CH

VL

Heavy chain

SS SS Whole antibody

SS

CL

Fab fragment

Antigen binding site

VH VL CL CH

Antibodies
mRNA RT-PCR cDNA Cloning Ampr Promoter VH domain coding region Fv fragment expression vector Expression

SS

Light chain

VH VL

Antigen binding site

4102 bp

VL domain coding region Fv fragment

ROP

Antibody fragment
Fv fragment or Fab fragment mixed with protein of interest Complex separated from free antibody or target protein using gel filtration Proceed with crystallisation trials

A difficult case-cytochrome c oxidase

Cytochrome c oxidase from P. denitrificans. Initially resisted crystallisation. Whole complex unstable in many detergents Addition of an antibody fragment facilitated the formation of protein-protein contacts.

Cytochrome c oxidase crystal packing

Antibody fragments
Time-consuming, risky and expensive Good option if crystals are available, extend the hydrophilic surface and obtain better diffracting crystal Rod McKinnons gp: KcsA potassium channel ClC chloride channel

Single conformation

WT LacY did not crystallise Cys154Gly mutant trapped protein in a single conformation

LacY structure

Summary
Membrane protein structural studies have made significant steps forward Rational screening Detergent screening Antibody fragment Lipidic cubic phase Single conformation Know your protein (or get a collaborator who does) Think hard and work hard

Acknowledgements