The importance of pre-crystallization screening of membrane proteins - and how to do it

The importance of pre-crystallization screening of membrane proteins - and how to do it

I don’t need to tell you that membrane proteins present unique challenges.

The prevalence of membrane proteins in organisms, their excellent potential as drug targets, and the relatively low number of solved structures is woefully clear to anybody working in this vital field.

But progress is being made and establishing methods to predict the formation of good crystals is playing a key role.

The importance of pre-crystallization screening

Growing well-ordered crystals which exhibit suitable diffraction to achieve high-resolution structures is known to be a major hurdle in the hunt for more membrane protein structures. And good screening has been shown to increase the success of these efforts (1).

Pre-screening is used to determine that the sample is monodisperse, is present in sufficient quantity, and is stable in its protein-detergent complex (PDC).

Of all these factors, the stability of the PDC is found across the literature to be key. The link between a stable PDC and likelihood of obtaining high quality crystals is observed (1), and the choice of detergent is stated as crucial (2) – evidenced by statements that the wrong detergent can encourage aggregation in samples (3).

While there are a growing number of methods available to perform this screening, one review on the topic (4) comments; “It is probably correct to state that expression, purifcation, crystallization, and structure determination of integral membrane proteins still remain non-trivial endeavors…” – we tend to agree.

How to perform pre-crystallization screening

We know it’s important, but how should you carry out the screening?


GFP screening is a popular approach. This involves expressing the protein of interest with a GFP tag attached and using this to detect its presence. Detection was historically by UV-280 or SDS-PAGE, but the large amount of sample these methods need made them inconvenient. Fluorescence SEC has become a more popular choice and adds the benefit of giving an indication of size as well as quantity (5).

This choice is great for early detection as even whole lysates can be tested, and the well-established fluorescence detection offers low detection limits of around 10 ng.

However, it should be noted that the GFP must be folded in the cytoplasm to do so correctly and fluoresce as required (6; 2). This means the membrane protein must have a cytoplasmic C-terminus available for attachment.


SEC-MALLS (size exclusion chromatography – multi angle laser light scattering) can be used to determine a range of features simultaneously; protein concentration, detergent concentration and protein molecular weight. This does offer more information on which to make decisions, but some studies using this approach note that it needs high concentrations to give reliable results (7).

CPM Assay

Many dye assays are unsuitable for membrane proteins as the presence of detergents increases background fluorescence levels, but CPM has been developed specifically to work with this class. The dye is only fluorescent upon binding cysteine residues, which only become available after heat-induced unfolding. This allows different stabilizing methods (such as the addition of lipids, antibodies, or change of detergents) to be screened, and requires relatively small amounts of sample. It is noted that the optimal dye dilution should be determined individually for each protein tested (8).


Microfluidic Diffusional Sizing (MDS) can report size and concentration of a protein from as little as 50 ng. In an MDS instrument, such as the Fluidity One, the labelling is performed automatically for you, and is protein specific so the presence of detergents is not a problem.

With size and concentration reported from one 10 minute test, you can quickly get a picture of your sample and repeat readings over the course of an hour to observe changes and infer stability.


1. Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures. Yo Sonoda, Simon Newstead, Nien-Jen Hu, Yilmaz Alguel, Emmanuel Nji, Konstantinos Beis, Shoko Yashiro, Chiara Lee, James Leung, Alexander D. Cameron, Bernadette Byrne, So Iwata, David Drew. 1, s.l. : Structure, 2011, Vol. 19, pp. 17-25.

2. Overcoming the challenges of membrane protein crystallography. Elisabeth P Carpenter, Konstantinos Beis, Alexander D Cameron, So Iwata. 5, s.l. : Current Opinion in Structural Biology, 2008, Vol. 18, pp. 581-586.

3. An efficient strategy for high‐throughput expression screening of recombinant integral membrane proteins. Said Eshaghi Marie Hedrén, Marina Ignatushchenko, Abdel Nasser, Tove Hammarberg, Anders Thornell, Pär Nordlund. 3, s.l. : Protein Science, 2009, Vol. 14, pp. 676-683.

4. A pedestrian guide to membrane protein crystallization. Wiener, Michael C. 3, s.l. : Methods, 2004, Vol. 34, pp. 364-372.

5. Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins. Toshimitsu Kawate, Eric Gouaux. 4, s.l. : Structure, 2006, Vol. 14, pp. 673-681.

6. A scalable, GFP‐based pipeline for membrane protein overexpression screening and purification. David Drew, Dirk‐Jan Slotboom, Giulia Friso, Torsten Reda, Pierre Genevaux, Mikaela Rapp, Nadja M. Meindl‐Beinker, Wietske Lambert, Mirjam Lerch, Daniel O. Daley, Klaas‐Jan Van Wijk, Judy Hirst, Edmund Kunji, Jan‐Willem De Gier. 8, s.l. : Protein Science, 2009, Vol. 14, pp. 2011-2017.

7. Structure and oligomerization of the periplasmic domain of GspL from the type II secretion system of Pseudomonas aeruginosa. Aleksandra Fulara, Isabel Vandenberghe, Randy J. Read, Bart Devreese & Savvas N. Savvides. s.l. : Scientific Reports, 2018, Vol. 8.

8. Microscale Fluorescent Thermal Stability Assay for Membrane Proteins. Alexander I . Alexandrov, Mauro Mileni, Ellen Y.T. Chien, Michael A. Hanson, Raymond C.Stevens. 3, s.l. : Structure, 2008, Vol. 16, pp. 351-359.

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