by Peter Nollert
December 2, 2009 04:08
One of the general advice for storage of crystallization experiments is to store them 'in the dark' or in 'dim light'.
Since I started my crystallization career with light-sensitive proteins this made a lot of sense to me. I'm wondering though, if the advise also holds up for the crystallization of light in-sensitive protein samples.
Turns out that at least some form of light can alter the outcome of crystallization experiments. Let's look at light that gets absorbed by protein, mostly via the aromatic amino acids Tryptophan, Tyrosine and Phenylalanine. These residues absorb light in the range of 280 nm, which can give rise to photo-oxidation reactions. Tstsuo Okutsu has published on the topic of UV light effect on nucleation.
Okutsu, T. (2007) hotochemically-induced crystallization of protein
Journal of Photochemistry and Photobiology, 8(3), 143-155
These experiments were carried out by 'weak UV irradiation' and there really shouldn't be a need to protect one's protein crystallization experiments from UV light because (i) there's little UV light around in a typical lab setting and (ii) most crystallization containers have poor UV transmission. Illuminating your samples with a strong UV source (i.e. UV microscope) is different though, and I'd like to hear from people what their experience is in this respect.
So, what about all the other light? Very strong pulses of light can apparently generate productive protein crystal nuclei. Check out this report where the effect of femto-second laser light on the formation of crystals of the membrane protein AcrB, ADA (adenosine deaminase) and human triosephosphate isomerase (TIM) was investigated. AcrB crystallization was dramatically improved. An ordinary laser pointer won't do the job, unfortunately.
Advanced protein crystal nucleation tool.
Finally, some of the formulations that are used in sparse matrix crystallization screening experiments are light sensitive and react over time. Hence the advice to store solutions containing Imidazol, MES buffer and Ammonium Iodide in dark containers. Once the photochemistry sets in, things become rather messy. That's not to say this is bad, since aggregated protein may serve as a nucleant. But light exposure is a factor in protein crystallization that should be carefully controlled.
Myth not busted.
Peter