Crystallised scorpion: Empirical emperor

A protein fit for a king

The first successful crystallization and X-ray diffraction study of enormous oxygen-transport protein, hemocyanin, from the emperor scorpion have been carried out.

One of the biggest scorpions in the world, the emperor scorpion, Pandinus imperator, also uses one of the biggest proteins, namely hemocyanin. This blood pigment, comprises a complex of 24 subunits thus rendering it comparable in size to the cell's protein-manufacturing unit itself the ribosome and even to some small viruses. Now, a team at the Johannes Gutenberg University Mainz in Germany have successfully grown crystals of this creature's hemocyanin and used X-ray crystallography to obtain a precise structure for the complex, building on the much lower resolution work with cryo-electron microscopy. The study will allow deeper understanding of the form and the function of hemocyanin.

Blue blood

Hemocyanins are well known as having remarkable size relative to other proteins. They are respiratory proteins used in oxygen transport in the blood of arthropods, such as scorpions as well as molluscs. Their mode of binding involves the chelation of an oxygen molecule by two copper centres, which contrasts with the human respiratory counterpart to hemocyanin, haemoglobin, which binds oxygen through an iron centre.

What makes hemocyanin even more intriguing is that depending on which animal species is being studied, the protein might use up to 160 oxygen-binding sites within a single protein complex and all of these must somehow communicate and coordinate the binding, transport, and release of oxygen by the blood. Chemists and materials scientists are very keen to mimic this kind of natural co-operativity for nanotechnology applications, such as molecular switches. The latest structural studies on hemocyanin may shed new light on how this co-operativity arises, taking us a step closer to understanding the processes involved in detail and so hinting out how it might be emulated in synthetic systems or how natural systems might be adapted for novel technological applications.

Elmar Jaenicke took the first decisive steps in the hemocynanin study by obtaining the first crystals of the protein complex from the P. imperator. The crystallization was, as if often the case for diffraction studies, largely a game of chance Jaenicke says. He explains that the crystallization process depended on pH, the salinity of the solution, temperature and various other factors. "The decisive step is always crystal nucleation," Jaenicke says. It can take months or even years to occur and so requires a great deal of patience. As many a frustrated crystallographer knows, this is the reason why essentially only a handful of molecular structures for very large protein complexes have so far been despite global effort.

Jaenicke and his colleagues obtained a mid-resolution (6.5 ångströms) structure early on and were able to discern secondary structure, such as the presence of alpha-helices. "This was our starting point and now we can already see parts of the active site of the molecule," he says. "With further improvements to our crystals, we are well on our way to achieving an atomic resolution that is not possible with any other method." According to Jaenicke, the oxygen binding protein from the emperor scorpion would then be one of the five largest structures to have been deciphered using X-ray crystallography so far. The X-ray work on the ribosome itself, of course, was rewarded with the 2009 Nobel Prize in Chemistry, which went to Venkatraman Ramakrishnan, Thomas A. Steitz and Ada Yonath.

The new rotating anode x-ray generator used at the Institute of Molecular Biophysics is, the researchers say, ideal for determination of the structure of such giant protein molecules because it produces focused X-ray beams with an intensity comparable to that of the beamlines at second-generation synchrotron facilities.

"The next step we are currently working on is to improve the crystals in order to ultimately make them diffract to atomic resolution (i.e. better than 3 A)," Jaenicke told SpectroscopyNOW. "This is currently done in the lab. Indeed crystals are improving, but we have not reached the 3A yet."