Protein Crystallization Hits

Every attendee received this SLAS2012 pin 

Cheers,

Peter

This week I flew down to San Diego to attend the protein purification sessions at PepTalk . The part I was most interested in was an inaugural session, called 'Higher Throughput Protein Purification" (not going to contemplate the 'higher than what?' issue). There were contributions from the fields of automation, particularly challenging targets, sample management and process development.  Interestingly, there were several common threads that the presenters discussed:

• Importance of using multiple constructs in purification and crystallization
• The one-size-fits-all approach is failing more often now with targets becoming more challenging
• Protein purification requires scouting on the level of lysis, purification and scale-up.

Portion of my 2012 PepTalk program on 'higher throughput protein purification'.

Clearly, our game is becoming more difficult on the side of protein purification, requiring new techniques and methods for attacking complex targets. 

Cheers,

Peter

Below is a slide presentation on protein crystallization that I am planning to show at PepTalk next week (Jan 9th, 2011, Sunday 3 pm in the Hotel Del Coronado, San Diego as part of the PepTalk Protein Science Week). The intention is to introduce workshop attendees to the topic of protein crystallization. 

If you think there's an important 'facette' missing, let me know now - I still have time to update my slide deck.

All the best,

Peter

Protein Crystallizers know this simple rule: for crystallization to occur protein samples should be pure, homogenous and the protein be in a stable and non-aggregated state. These requirements can often be met by applying standard purification and concentration procedures to soluble protein targets. This is one of the reasons for the productivity of high-throughput structure genomics-type efforts. The buffer type, pH and salt are typically standard systems - such as 100 mM NaCl, 50 mM Tris-HCl pH7.5 - that rarely requires optimization for a particular protein target.

These requirements are also valid for membrane proteins, but achieving them often requires a lot more effort. A typical parameter to evaluate for membrane proteins is the detergent type and its concentration. Such a crystallization pre-screen can be done sometimes even before starting with the purification and, the procedure applied is called 'detergent exchange'. It turns out that there's a handful of methods available to carry out such detergent exchanges, for instance, using size exclusion chromatography, simple dilution or ultracentrifugation. The logic goes like this: once the membrane protein aggregates in the newly tested detergent solution, it can be detected as an extra peak, running with the void volume in the sizing chromatogram, as an increase in turbidity, or as a decrease in protein concentration in the supernatant of an ultracentrifuge tube.

Anybody who has concentrated a protein sample using a simple MWCO filter realizes, that filtration provides a good means to separate aggregated from non-aggregated protein. Indeed, GE Healthcare's multi trap purification filter kit utilizes  this feature for purification scouting of His-tagged proteins. Essentially, the protein is bound to a resin and eluted by filtration through a MWCO filter. The conditions that yields most protein in the filtrate wins. Michael Wiener's membrane protein group has given this concept an interesting twist. They describe in this paper:

an adaptation of the filtration concept to detergent screening and have devised a differential filtration assay that allows to identify those detergents that render a particular membrane protein target 'well behaved' and hence, more crystallizable. How does this work?

Instead of using the individual filtration devices, two 96-well filter plates of different MWCO (the use of two different MWCO sizes allows to distinguish between stability and size) are used sequentially. 94 different detergents can be used in one go. Some people may consider testing with 94 different detergents overkill, but I'm with Michael in his 'knock em dead' approach. However, using fewer detergents and spin-filters may prove more practicable in many labs. In fact, this useful review on membrane protein crystallization parameters:

Newstead, S., Ferrandon, S., & Iwata, S. (2008). Rationalizing α-helical membrane protein crystallization Protein Science, 17 (3), 466-472 DOI: 10.1110/ps.073263108

can be used to argue that by just testing 8 instead of 94 different detergents one can cover more than 80% of the 'detergent landscape'. The table below shows the ranking of detergents that have provided most membrane protein crystal structures. Note that followers of the Pareto Principle (80% of the bang for 20% of the buck) will do with a set of only 8 detergetns or filter tests.

Table: Which detergents to use for membrane proteins? Shown are detergent types and occurrences in successful membrane crystallization screens. Data from supplemental info to Newstead et al., 2008 paper, kindly provided by Simon Newstead. Note 1: Am I seeing this right that HEGA-10 is not included in the set of 94 used by Wiener?; Note 2: adhering to this table will increase investigator bias.

When I saw Michael presenting his differential filtratio assay at the NIH Roadmap Meeting in March 2009 I was particularly excited by these features:

(i) the low sample amounts required (only 10 ug protein / well to create sufficient signal in the dot-blot / western assay) and,

(ii) the type of data that can be pulled out of this assay.

It has taken me a while to appreciate the utility of the 'size stability quad plot'. The gist is that the membrane proteins can be grouped into cohorts that are formed by the quandrants as defined by stability and size. This sorting of detergents helps to predict those detergent species that should work best for crystallization.

And that's exactly the type of result you're expecting from a crystallization pre-screen.

All the best,

Peter

Over the years there have been a number of Nobel Prizes awarded to scientists that have used their protein crystallization skills to provide unprecedented insight, usually at atomic resolution, into important biological processes. In appreciation of their contribution I had put these 12 crystallization heroes into the Protein Crystallizers Hall of Fame with the crystallization and structure determination of these proteins: Ribosome (2009), Water & Ion Channels (2003), RNA Polymerase (2006), Photosynthetic Reaction Centre (1988), Ion transporters (1997).

Which are the possible Protein Crystallization-related Nobel Prizes this year? What do you think - which of these target areas would you consider for a 2010 Nobel Prize (Chemistry or Medicine)?

Or here if you prefer to vote via your LinkedIn account: 

There are plenty of reasons to select these protein structures since they have provided useful insight into biological function and should therefore be worthy a 2010 Nobel Prize:

Let's keep in mind though, that the Nobel Committee may take a break from structural biology in 2010 and focus on these areas that come to mind:

□ Discoveries of pluripotent stem cells / dentritic cells

□ Technologies: human genomics, sequencing, DNA microarrays

□ Pathways and Drugs: Leptin, DNA metallo intercalators

Now, with the 2010 Nobel Prize announcements coming up next week I'm keeping my fingers crossed for yet another award for this fine scientific craft. The announcements are expected to be made on

Regardless of the outcome next week, it's definitely an occasion that's worth getting a bottle of champagne out of fridge!

Cheers,

Peter