Protein Crystallization Hits

One of our scientists - Tim Craig - has turned his attention to a fundamental barrier that needs to be overcome in any membrane protein research project: functional extraction of membrane proteins from their biological membrane.  Every membrane protein target needs to be matched with a particular detergent reagent or a set of detergents. Since Tim was working with fluorescently labeled membrane proteins, he applied an experimental approach that has gained popularity in recent years: FSEC. That is: Fluorescence-Detection Size-Exclusion Chromatography; this is primarily a pre-crystallization method for membrane proteins that has been developed in Eric Gouaux' lab (see references below). 

This is how it works: solubilize a membrane with a particular detergent and apply the resulting sample to size exclusion chromatography with a benign detergent (i.e. dodecyl maltoside) in the mobile phase. Repeat with as many detergents as possible. Inspect the traces for symmetry of the (largest) peak and absence of signal in the void volume.  Pick the best detergent(s) to establish a purification and crystallization procedure. The neat trick here is that such crystallization-relevant information can be obtained at a very early stage in your membrane protein project, for instance once you can prepare membranes. This methodology solves the hen-and-egg problem (need to have a detergent in order to be able to purify a particular membrane protein, need to have a pure membrane protein sample in order to test which detergent is compatible) and is simple to automate with standard HPLC instrumentation.   

Since preparing many different detergents is a laborious process we thought we'd pack them into a kit. And this is it, the Wizard: TIME screen Use the Wizard: TIME screen to conveniently determine the detergent that successfully extracts your target membrane protein from a membrane preparation.

In order to maximize both, extraction efficiency and membrane protein stabilization, each of the 84 different detergent reagent is formulated at 2% (w/v) together with the co-detergent cholesterolhemisuccinate. Positive and negative controls specifically for FSEC make this screen a very convenient tool for fast, simple and information-rich membrane protein extraction assessment. 

The complete listing of detergents in the Wizard: TIME screen are available here.

Our recommended Wizard: TIME screen sample treatment protocol:

1. Mix 125μL of your membrane preparation with the contents of each well in a fresh 96-well plate (membranes should be prepared at a concentration that allows for good signal:noise in the analytical assay of your choice). 

2. Incubate the mixture to allow for protein extraction (different incubation times and temperatures will influence extraction efficiency, however typically 1 hour at 4°C can serve as a good starting point for extraction optimization). 

3. Separate extracted material from debris by ultracentrifugation at >100,000 x g for 20 minutes using a small volume rotor or by filtration using a filter. Keep either the ultracentrifugation supernatant or the flitration flow through and

4. Analyze samples with an analytical fluorescence detection SEC-HPLC.  

Identification of suitable detergent for membrane protein detergent extraction with the Wizard: TIME screen using FSEC. A) LDAO produces large, in-homogenous membrane protein particles, a sizeable fraction of which running in the ‘void’ volume. B) CYGLU-4  extracted membrane protein runs mainly as a symmetric single peak. 

 Here is a link to order info for the Wizard: TIME screen. More details on the FSEC protocols are described here: 

Hattori M, Hibbs RE, & Gouaux E (2012). A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure (London, England : 1993), 20 (8), 1293-9 PMID: 22884106

 KAWATE, T., & GOUAUX, E. (2006). Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins Structure, 14 (4), 673-681 DOI: 10.1016/j.str.2006.01.013 

Final tip for dealing with detergent-solubilized membrane protein samples: no bubbles!

Peter

It can be hard keeping up with the fast progress in membrane protein structural biology. I realize that is especially true on the fringes of my particular area of interest. The sheer amount of progress is mindboggling. Since you can't read all new publications - what are you missing out on? What are new developments that are important to your work? How do you separate the noise from the new methods that can have a high impact in your lab projects? I have realized that reading reviews doesn't cut it any more. Scientific meetings are a great for this purpose, but they're not exactly cheap and happen infrequently.
I have learned to appreciate the space between written reviews and scientific meetings: webinars and podcasts on topics that interest me.
For instance, here's a Podcast on 'Recent advances in structural biology methods' by Liz Carpenter and Richard Neutze.
Here at Emerald Bio we're not only consumers, but also producers of such new media. For instance, Jim Fairman will be presenting on Tools for the Expression and Crystallization of Membrane Proteins
Monday, November 12, 2012, 4:00pm – 5:00pm EST = 2 pm
Participate by signing up here

Enjoy,
Peter

If only we would not have to ‘waste’ so much protein sample for mind-numbing trial and error crystallization experiments. Aside from clever pre-crystallization screening approaches, there are now several reports that indicate that we may start to get a handle to more rational approaches. This is the first short review of such a report towards shortening the path from protein sample to crystals.

The observation is that ‘Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success’. A post-mortem analysis of purification and crystallization data has revealed quantitatively what many of us have picked up at the bench: proteins that purify well are easy to crystallize.

Choi R, Kelley A, Leibly D, Hewitt SN, Napuli A, & Van Voorhis W (2011). Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success. Acta crystallographica. Section F, Structural biology and crystallization communications, 67 (Pt 9), 998-1005 PMID: 21904040

The authors of this paper however have taken this observation as a starting point and have devised a low cost IMAC high-throughput protocol (using multichannel pipettors, affinity beads and filter plates; the procedure is described in exquisite detail; thank you very much!). They applied this protocol to more than 4330 proteins (SSGCID effort) to mine IMAC recovery data for rules and find that they determined more than twice as many structures of those proteins that showed a high IMAC recovery, as compared to those with a low IMAC recovery.

Factor 2. 

Not bad.

Protein researchers of the world: here's your chance to make a lasting impact with information, ideas and comments on the topic of future disruptive proteomics technologies. The NIH has an RFI (Request for Information, NOT-RM-12-015) out, asking you to provide input in to how to accelerate research in disruptive proteomics technologies.

There are simple online forms where you can provide your anonymous feedback on the following topics:

1. MS-based comprehensive protein identification and quantification. Realistic goals and associated challenges for orders-of-magnitude improvements in dynamic range, sensitivity, throughput or cost. Specific areas of instrumentation (e.g., source design/analyzer geometry, coupling with other instrumentation, etc.) more likely to yield disruptive improvements.
2. Non-MS-based comprehensive protein identification and quantification. Opportunities and challenges for other technologies that could in the near or far term approach/exceed MS-based methods with respect to: accuracy, dynamic range, throughput, and cost in analyzing proteomes.
3. Potential benefits and challenges in incorporation of informatics approaches and/or integration of large protein datasets into the development of the proteomic technologies.
4. Potential important impacts of proteomics technology breakthroughs in basic, discovery and translational biomedical research.

This unique opportunity is available until March 26.

          Quick! Disruptive proteomics technologies. The NIH wants to know what you're thinking.

What are you going to get out of this? No immediate grant awards. But your information may be included in planning  future grant funding opportunities that seek to fund disruptive technologies that have occurred in DNA sequencing technologies in the past few years.

And that impact may be larger than any of the papers you have ever published.

This week there are two fascinating stories in Nature Methods each giving us a glimpse of what structural biology might look like in a decade or so. Both papers describe a technical tour de force, shooting jets of micro crystals into the beam of a X-ray free electron laser and collecting X-ray diffraction images.

The first report utilizes recombinant protein (TbCatB) crystals that are grown in Sf9 insect cells. Yes, that's right: protein crystals grown in vivo, no crystallization setups necessary here. 

Koopmann, R., Cupelli, K., Redecke, L., Nass, K., DePonte, D., White, T., Stellato, F., Rehders, D., Liang, M., Andreasson, J., Aquila, A., Bajt, S., Barthelmess, M., Barty, A., Bogan, M., Bostedt, C., Boutet, S., Bozek, J., Caleman, C., Coppola, N., Davidsson, J., Doak, R., Ekeberg, T., Epp, S., Erk, B., Fleckenstein, H., Foucar, L., Graafsma, H., Gumprecht, L., Hajdu, J., Hampton, C., Hartmann, A., Hartmann, R., Hauser, G., Hirsemann, H., Holl, P., Hunter, M., Kassemeyer, S., Kirian, R., Lomb, L., Maia, F., Kimmel, N., Martin, A., Messerschmidt, M., Reich, C., Rolles, D., Rudek, B., Rudenko, A., Schlichting, I., Schulz, J., Seibert, M., Shoeman, R., Sierra, R., Soltau, H., Stern, S., Strüder, L., Timneanu, N., Ullrich, J., Wang, X., Weidenspointner, G., Weierstall, U., Williams, G., Wunderer, C., Fromme, P., Spence, J., Stehle, T., Chapman, H., Betzel, C., & Duszenko, M. (2012). In vivo protein crystallization opens new routes in structural biology Nature Methods, 9 (3), 259-262 DOI: 10.1038/nmeth.1859 

The second paper describes a similar experiment, carried out with small crystals of the Blastochloris viridis photosynthetic reaction center grown within lipidic phases. The resulting images actually resemble conventional X-ray diffraction images with proper Bragg spots, good enough to build a somewhat meager 8.2 Å resolution electron density map.

Johansson LC, Arnlund D, White TA, Katona G, Deponte DP, Weierstall U, Doak RB, Shoeman RL, Lomb L, Malmerberg E, Davidsson J, Nass K, Liang M, Andreasson J, Aquila A, Bajt S, Barthelmess M, Barty A, Bogan MJ, Bostedt C, Bozek JD, Caleman C, Coffee R, Coppola N, Ekeberg T, Epp SW, Erk B, Fleckenstein H, Foucar L, Graafsma H, Gumprecht L, Hajdu J, Hampton CY, Hartmann R, Hartmann A, Hauser G, Hirsemann H, Holl P, Hunter MS, Kassemeyer S, Kimmel N, Kirian RA, Maia FR, Marchesini S, Martin AV, Reich C, Rolles D, Rudek B, Rudenko A, Schlichting I, Schulz J, Seibert MM, Sierra RG, Soltau H, Starodub D, Stellato F, Stern S, Strüder L, Timneanu N, Ullrich J, Wahlgren WY, Wang X, Weidenspointner G, Wunderer C, Fromme P, Chapman HN, Spence JC, & Neutze R (2012). Lipidic phase membrane protein serial femtosecond crystallography. Nature methods PMID: 22286383

Granted, all of this is currently in the proof-of-concept stage - no actual high resolution structure determined yet - but this is how new exciting breakthrough technologies often start out.  I'm wondering how long it will take for these technologies to mature to a state where they produce useful resolution structures and when they will become applicable to 'the rest of us'. Ten years, mid of the century maybe?

No protein crystallization setups, no crystal harvest, no cryo. X-FEL kills the crystallization champ.

This might change our game quite a bit.

Cheers,

Peter