INSANE IN THE MEMBRANE: HOW TO OVERCOME PROBLEMS WHEN STUDYING MEMBRANE PROTEINS.

Approximately 30% of the genes in the human genome code for membrane proteins, and yet we know relatively little about these molecules. Less than 1% of structures currently in the protein data bank correspond to membrane proteins, but this is not because a lack of interest but because working with these molecules is challenging and difficult. I would even dare to say that scientists that work with membrane proteins are unique, they are the bravest and more patient of us all.

Since many membrane proteins sit at the surface of cells, they are readily available to small molecule drugs circulating in the blood, thus they are important pharmaceutical targets. It is therefore not surprising that over 60% of small molecule drugs bind to membrane proteins.

Membrane proteins are unfortunately notoriously difficult to handle and study since they are designed to sit within the hydrophobic environment of the lipid bilayer. Common issues include:

• -        Expression
• -        Extraction
• -        Solubilization
• -        Purification
• -        Biophysical studies

RELIEVING THE COMPLEXITY OF STUDYING A MEMBRANE COMPLEX 

Working with membrane proteins is still based on trials and errors. From cloning and expression (choosing the right microorganism, what tags to use, setting conditions for overexpression), solubilization (testing different detergents) and reconstitution in artificial membranes, but it also depends on choosing the right tools, which can greatly ease your job, save a lot of time and money and more importantly that can retrieve valuable and precise information as in the case of biophysical techniques used to study their structure and function (molecule binding affinity and specificity, interaction with other molecules, etc).

In a beautiful study published on 2014 on JBC, Sabine Ebbensperger et al.¹ from Goethe University showed how these problems were overcome in the study of the membrane complex TAP.

THE TAP COMPLEX

When I was a kid I had chickenpox and it was a mess. I was so unlucky that the virus infected my brain and caused me encephalitis leaving me with some vision problems. Anyway, at the end it was me who won the battle and nowadays I still think how fascinating it is to overcome a viral infection. But how does that happen? Well, the immune system can destroy cells that are infected with viruses or otherwise damaged, when cytotoxic T-lymphocytes recognize antigenic peptides presented by MHC class I molecules (MHC I) on the cell surface. But how on earth does an antigenic peptide, coming from viruses or damaged cells, end up in the cell surface?

Well long story told short, antigenic peptides are degraded by the proteasome in the cytosol while MHC I molecules fold and assemble within the endoplasmic reticulum (ER). As a consequence it is necessary to translocate peptides from the cytosol into the ER lumen and this function is performed by the transporter associated with antigen processing (TAP) which uses the energy from ATP. TAP also acts as a scaffold for the final stage of MHCI assembly, i.e. peptide binding.

TURNING OFF THE TAP… HOW CLEVER CAN A VIRUS BE

TAP is a heterodimer formed by the subunits TAP1 and TAP2. Its membrane domains form the peptide-binding site, whereas ATP hydrolysis occurs at the nucleotide-binding domain (NBD), which energizes peptide translocation the ER lumen by membrane domains.

But of course, virus are smart and have evolved proteins that interfere with this pathway!!!!...... and…….. TAP is one viral target that is exploited for example by the herpes simple virus (HSV) ICP47 small protein. ICP47 inhibits peptide binding to TAP, but does not affect ATP binding. Yet ICP47 does not behave like a normal peptide as it is not translocated across the membrane and it remains associated with TAP.

THE CHALLENGE: TO STUDY THE TAP COMPLEX

PURIFICATION

In this work, TAP was expressed in yeast, with each subunit affinity tagged (His₁₀ and StrepII) to facilitate purification, and fusioned to fluorescent protein tags which allow direct visualization of the target during expression, solubilization and purification and can speed up the optimization of these processes. Way to go.

During the solubilization stage, membrane proteins are extracted from the lipid membrane to an aqueous environment by the use of detergents, which disintegrate the lipid bilayer while incorporating lipids and proteins in detergent micelles. Then, proteins re ready to be purified.

A great approach of this work and that makes a huge difference is that the TAP complex was then purified by tandem affinity of its subunits and detected by Multi Color Fluorescence Size Exclusion Chromatography (MC-FSEC), that as FSEC is used to enable tracking of individual subunits through expression, solubilization and purification steps. But what makes it better than FSEC though is its ability to detect multiple subunits of membrane protein complexes, such as TAP1 and TAP2, simultaneously and to analyse their behaviour in numerous conditions. It allows rapid assessment of the correct assembly and stoichiometry under the conditions tested and as the authors illustrated, it is suitable for the study of an hetero-oligomeric membrane protein complex.

RECONSTITUTION OF TAP IN NANODISCS

Reincorporation of purified membrane proteins into an artificial membrane continue to be crucial in studying the function and structure of these molecules. The necessity for reconstitution arises because many membrane proteins express their full activity only when correctly oriented and inserted in a lipid bilayer. 

Nanodiscs (ND) are self-assembled discoidal fragments of lipid bilayers 8-16 nm in diameter, stabilized in solution by two amphipathic helical scaffold proteins.

Now, nanodiscs provide several key advantages:

1.     Small size compared to liposomes

2.     Stoichiometry and composition of membrane protein and lipids can be controlled precisely.

3.     Substrate, ligand and protein interactions can be studied in close-to-native environment with access to both sides of the membrane protein complex.

TAP was successfully reconstituted in nanodiscs, with TAP1 and TAP2 subunits in nanodiscs detected by MC-FSEC. Moreover, the reconstitution procedure maintained TAP function (peptide binding and ATP hydrolysis) and gave important information like the annular lipid belt surrounding the TAP complex in nanodiscs is essential for high affinity IPCP47-TAP interaction.

PEPTIDE BINDING ANALYSIS BY MICROSCALE THERMOPHORESIS (MST)

Although biophysical methods have been successfully applied to an array of soluble protein targets they have failed in one way or another when applied to membrane proteins.

MST reports on a direct ligand-protein interaction and it is based on the differential movement of free fluorescent peptides versus and bound fluorescent peptides in a temperature gradient, induced by an infrared laser.

Accordingly, the change in fluorescence was used to reflect the concentration of peptide-TAP complexes. This technique was used to analyze peptide binding of TAP reconstituted in nanodiscs. More specifically it retrieved information about:

• -      Peptide binding affinity of TAP (Equilibrium dissociation constants K_D for nanodiscs-reconstituted TAP as well as detergent-solubilized TAP were obtained).
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• -        Specificity of peptide-binding.
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• -        Mode of action of the viral inhibitor ICP47 (using competition microscale thermophoresis it was demonstrated that ICP47 competes with peptides for the TAP binding pocket).

All in all, the combination of nanodiscs, MC-FSEC and MST worked very well in the study of a membrane complex. It certainly eased the job and showed that good information can be obtained from a difficult study model, if attention to the available technologies is paid.

MST holds promises to the study of membrane proteins and complexes and for the analysis of pharmaceutical-relevant molecules directed towards them.

1.     Eggensperger S, Fisette O, Parcej D, Schäfer LV, Tampé R. J Biol Chem. 2014 Nov    28;289(48):33098-108.