Protein Crystallization Hits

I'd estimate that more than half of all protein crystallization experiments employ protein that has a poly-Histidine tag. These are usually hexahistidine tags at the N-terminus or at the C-terminus of a target protein to aid and simplify their affinity purification with metal chelate chromatography (IMAC). Since it is cumbersome and costly to remove such tags, many crystallographers keep the extra amino acids that come with the tag and subject the protein to crystallization trials without His-tag removal.

What is to be done if N-terminal His-tagged proteins with a TEV-proteolytic site don't yield crystals or the grow crystals with poor X-ray diffraction quality only? Dong et al. have come up with an ingenious protocol, in-situ proteolysis.

Simply put, crystallization experiments are prepared in the presence of trace amounts of a protease, for example chymotrypsin. This works for target proteins that have a recoginition site for TEV-protease, such as in MGSSHHHHHHSSGRENLYFQG or MGSSHHHHHHSSGRENLYFQGH where chymotrypsin cleaves at Tyr and Phe.

Since the authors were affiliated with high-throughput crystallography operations they were able to process many targets with their new in-situ proteolysis protocol. Altogether they subjected 55 different bacterial proteins (20 had previously failed to crystallize and crystals of the remaining 35 were no good) to the in-situ proteolysis protocol and ended up with 9 rescued targets. Not bad, converting 55 crystallization recalcitrant proteins into well diffracting crystals, yielding 9 new structures.

This is their in situ proteolysis protocol:

α-chymotrypsin (SigmaC3142), dissolved at a concentration of 2 mg/ml in a solution containing 1 mM HCL and 2 mM CaCl2, was added to the purified protein on ice immediately prior to crystallization trials at a ratio of 1μg chymotrypsin per 100 μg of histidine-tagged protein, dissolved at 10-20 mg/ml in 10 mM Hepes, pH 7.5 and 500 mM NaCl. Crystallization was performed in sitting drops at room temperature, adding 0.5 μl of the protease/protein mixture to 0.5 μl of the precipitant. Crystallization trials were set p immediately, without assessing the efficacy of the proteolysis, without stopping the roteolysis reaction, and without purification of any proteolyzed fragments.

In the supplemental material the group describes several examples of target proteins that crystallized only in the presence of the proteases or cases where the X-ray diffraction quality was improved. The description of the crystallization stories in the supplemental material make for a great read.

Here's a summary of the different cases encountered:

• SCO6256 failed to crystallize and in the presence of chymotrypsin two different crystal forms were obtained, one of them diffracting well enough for phasing and 2.5 A structure determination
• SCO4942 did not yield crystals with conventional crystallization screening efforts but yielded 2.8A-diffracting crystals after subjecting to chymotrypsin treatment
• NE2398 crystals grew to reasonable size but did not diffract. When re-screened in the presence of chymotrypsin, 1.8A X-ray diffracting crystals grew.
• ATU0899 grew as small needle cluster crystals that did diffract to only 5A; after in-situ proteolysis crystals diffracted to 1.8A
• ATU2452 grew small needle clusters (sea urchins) and the chymotrypsin treated protein grew as nice diamond-shaped crystals that diffracted to 2.5A
• ATU0870 grew as multiple crystals with his-tag and tag removed diffracted to 2.5A. When crystallized in presence of chymotrpysin, 1.95A X-ray diffraction data was obtained.
• HP0029 grew as thin needles as his-tagged protein, best diffraction was 3.2A; removal of his-tag using TEV failed; with chymotrypsin treatment 1.47A data was recorded and structure determined
• ATU0299 grew as un-optimizable needles that diffracted to 1.8 A when crystallized as a mix with chymotrypsin
• ATU0434 crystallized in 8 cocktails but diffracted only to 3.5A; with chymotrypsin treatment crystals did not get any better, but with trypsin treatment crytsals grown diffracted to 2.3A.

Interestingly, the authors revisit the structures they obtained with the in-situ chymotrypsin treatment and they make hindsight guesses as to why the crystallization of the untreated proteins did not work. For several cases 'it was difficult to ascribe a mechanism by which proteolysis facilitated crystallization in most cases' but there were several exceptions. One in particular showed that extended N-terminus would have disrupted packing in the obtained crystal form.

Check out the optimized 96-formulation crystallization screen to be used for such in-situ proteolysis experiments.

When you're dealing with an otherwise hopless case, a 16% rescue rate is not that bad.

Cheers,
Peter

P.S. just found the update to this paper, with better statistics. May post on this later.