On crystallising the "high-hunging fruits" - collection of unusual strategies for protein crystallisation

For long has it been believed that the process of macromolecular crystallisation is stochastic - one is completely at mercy of the target protein.

The development of modern techniques are about to change this however, by directly addressing the inherent "crystallisability" of the proteins. Furthermore, there has been developments from other direction which I believe is noteworthy. Here I will make a collection of publications and notes about several unusual strategies in protein crystallisation pridominantly as a note for myself, but I hope it will be of use to whoever forwarded here by search engines. I will attempt the contradictory - keep it brief while maintaining the core information that distinguishes each of them.

Surface entropy reduction strategy
This method will directly address the crystallisability of the protein by creating a crystal contact patch on the surface of the protein. Crystallisation may be regarded as a chemical reaction where the lower the Gibb's free is, the better it proceeds. Some amino acids such as Glumate and Lysine will provide negative conformational entropy if they are to be stabilized during crystallisation. Now, negative of a negative will contribute positively to the overall Gibb's free energy.

This approach is my personal boom. This will be relevant and applicable in many situations because the majority of the difficult protein in progress will be cytoplasmic (they would have been chosen on an assumption that cytoplasmic protein will be easier to crystallise). Application of this technique to a membrane protein has shown success too.

Supply crystal contact sites
This approach is somewhat akin to the method above in the sense that crystal contact site is artificially introduced. This may be applicable to both cytoplasmic and membrane proteins.

For cytoplasmic proteins that are hard to "tame", it is an usual approach to add a tag such as MBP or GST to the protein to increase its solubility/stability. Usually the expression and purification is followed by the cleavage of the tag, but in this review, the authors discuss the approach to crystallisation with the tag intact. This has advantages such as shorter purification steps (no cleavage condition optimisation and separation) and additional source of phase information via molecular replacement. The drawback of potential flexibility between the tag and the target protein is addressed by opting for an inflexible linker comprising of polyalanine chain over the usual protease target sequence. The length of the linker is subject to optimisation.

Membrane proteins may be inherently less susceptible to crystallisation than cytoplasmic proteins. The solublisation of the membrane protein involves the use of detergents which forms vesicles whose radius is substantial relative to the dimension of the protein. The polar surface available as crystal contact site is hence potentially limited, and perhaps this also rationalise the more fragile nature of membrane protein crystals. In a recent structural report of highly anticipated GPCR - G-protein complex, the authors report the multitudes of techniques to overcome the problem. Llama nanobody was used to stabilise the flexible apo-form of the alpha subunit, but the interesting part for me is the incorporation of T4-lysozyme in between the transmembrane helices which served as crystallisation tag that sticks out of the vesicle and provide inter-protein contact areas.

Low gravity crystallisation
Low-gravity environment in the space has opened up possibilities for many novel experiments, as well as obtaining better macromolecular crystallisation. Those who can't afford such an expensive experimental set-up need not despair, however. A strong magnetic field that mimic the low-gravity environment has been shown to promote crystallisation of lysozyme. Sound excellent, though to what extend will this be practical is questionable. I personally have not seen any follow-ups to the New Scientist article above which is dated August 2007, but it could just have been me missing it.

Laser induced crystallisation
Exposing the crystallisation drop to circularly polarized light at 532nm has been reported to promote crystallisation. The effect to protein solubility has also been reported too.