Advances of Protein Crystallography

J. Hašek

Inst.of Biotechnology and Inst.of Macromolecular Chemistry,

Academy of Sciences of the Czech Republic, Praha, Czech Republic

hasekjh@seznam.cz

The General Assembly of the United Nations (UN), on its 121^st plenary meeting decided to proclaim 2014 as the International Year of Crystallography. The following text contains several quotes and sentences reproduced from the 66/284 General Assembly declaration:

“Recognizing the leading role of the International Union of Crystallography (IUCr), an adhering body of the International Council for Science (ICSU), in coordinating and promoting crystallographic activities at the international, regional and national levels around the world, ......

The humankind’s understanding of the material nature of our world is grounded, in particular, in our knowledge of crystallography. The impact of crystallography is present everywhere in our daily lives, in modern drug development, nanotechnology and biotechnology, and underpins the development of all new materials, from toothpaste to airplane components. Applications of crystallography are critical in addressing challenges such as diseases and environmental problems, by providing protein and small molecule structures suited for drug design essential for medicine and public health, as well as solutions for plant and soil contamination.

Persistent flow of Nobel prizes shows that the crystallography is still fertile ground for new and promising fundamental research.

Therefore, it encourages all Member States, the United Nations system and all other actors to take advantage of the International Year of Crystallography to promote actions at all levels aimed at increasing awareness among the public of the importance of crystallography and promoting widespread access to new knowledge and to crystallography activities.

It is also worth to commemorate the sixty-fifth anniversary of the founding of the International Union of Crystallography and the foundation of the journal Acta Crystallographica involving presently six volumes A,B,C,D,E,F devoted to specialized aspects of scientific research.”

Nobel prizes awarded for crystallography or in close connection to crystallography were already listed in a short review /1/. In this paper, we concentrate especially on the last development of the protein crystallography, i.e. structure determination of large biomolecular complexes in atomic resolution where the international appreciation seems to be the highest.

Figure 1 summarizes the counts of Nobel prizes by each ten year since 1901 (the year when the first Nobel prize was awarded). In comparison with the chart published in [1], one can see here marks of oversampling. Splitting the intervals to halves makes the profiles more humped but because we know the background of each event we can follow the causes of these humps in history.

The tremendous research on development physical methods of crystallography lasting more than 40 years in the period 1895-1940 was appreciated by seven Nobel prizes.

The high potential of X-ray crystallography for many important activities of mankind was fully recognized in the period 1960-1980 when a completely new and economically promising insight into the micro-world of molecules and molecular machines discovered completely new view on modern technologies. New discoveries opened completely new approach to molecular biology, revealed how molecular machines work and showed basic principles behind function of living organisms. These discoveries were rewarded with five Nobel prizes.

Recognition of high importance of crystallography induced large investments in the field, namely in development of technologies and crystallography methods. Thus, we can see four Nobel prizes in 1980-2000 for development of techniques and also four Nobel prizes for applications in chemistry and molecular biology mostly due to the dissemination of new methodologies and their accessibility over the whole world.

Finally, the recent development (13 years of this century) shows that X-ray crystallography still has a high potential for the future of mankind. Seven Nobel prizes awarded in the period 2003-2013 uniquely show high importance of the achievements. Particularly promising seems to be the statistics in the field of protein crystallography, where one can be fascinated by a new Nobel prize every second year.

Fig.1. Numbers of Nobel prizes closely related to applications of crystallography methods in chemistry and biology in ten years intervals from 1900-1910 until now. The last points count the Nobel prizes per the period 2001-2013

Why is protein crystallography regarded so important?

Protein crystallography showed us the world of living organisms at atomic resolution for the first time. This was necessary to understand and rationally regulate the processes important for life. It allowed formation of completely new fields of research and opened our eyes to understand a huge complexity of processes in living organisms. The illustrations in the appendix show some examples answering the questions concerning:

· exact conformation and the conformational changes of the largest known molecular complexes as e.g. ribosomes,

· determination of intermediate states necessary for understanding the biochemical reactions,

· design of completely new biomolecules showing far better performance than their counterparts present in nature,

· detailed elucidation of enzymatic function and design of new catalytic centers providing biochemical reactions allowing easy production of rare or beforehand unavailable chemical products,

· time resolved analysis of bio-chemical reactions, even if it is presently restricted mostly to processes that can be initiated or modulated by a pulse of electromagnetic radiation,

· analysis of intermolecular contacts, forming a base for intercellular communication and signaling between cells in the body. The following figure showing the supposed dimerization of receptors at the surface of natural-killer cells to accept the information whether to start or suppress the attacks against other cells in its neighborhood can serve as an example.

Figure 2. Comparison of dimerization modes of three mouse CTL proteins: NK receptor NKR-P1A (PDB code 3M9Z), NK receptor Klrg1(3FF9), and a ligand for NK receptor NKR-P1F: Clr-g (3RS1). Reproduced from [2].

Future applications of X-rays in structural biology?

The question about future applications of X-rays in structural biology can hardly be answered well. It seems that, after the huge progress due to better technologies (computers, advanced detectors, synchrotron sources of radiation) in the last twenty years, we need now to develop more sophisticated methods for sample preparation. Here are many points, which seem to be bottlenecks of the present research.

1. Exact structure determination of bio-macromolecular complexes in their native state. As a rule, the living entities form a special state of matter. The natural environment in living organisms where the most interesting biochemical reactions appear is not liquid, neither solid. It is molecularly overcrowded state of matter often with thixotropic properties, diffusion of molecules is overwhelmed by other transport mechanisms, intermolecular interactions do not correspond to what we can see in solutions, etc. Here, one can see a large space for new sophisticated procedures to prepare samples that could be inspected by X-ray crystallography. For example new methodic for controlled preparation of realistic multi-protein crystalline states allowing a systematic study of intermolecular contacts responsible namely for communication between cells, for immune response, for allergy, and for starting or modulation of processes in living organisms.

2. Inspection of detailed differences between similar supra-molecular complexes and their intermediate states important for life of different organisms. The final aim is for example to stop some process in harmful bacteria without influencing the human proteins or proteins in cohabiting microorganisms.

3. Time resolved studies. They are still in their beginnings. The problems here are not in the diffraction technique. Standard synchrotrons allow already much better time-resolution (~10 ns) then it is needed for biochemical reactions (µs-ms). Shortening of the radiation pulls to femtoseconds offered by X-FEL and some new laser facilities is harmful in this sense, because each pull destroys the sample. The future problem is in finding tools for immediate starting the reactions in the whole volume of our sample and in dealing with the multiconformational states regularly observed in the individual time sections.

The impact of the future research is expected in many areas of human life via molecular biology, biochemistry, chemistry, health care, drug design, agriculture, food industry, chemical industry, etc.

References

/1/ J. Hašek, International Appreciation of X-ray Crystallography. Materials Structure, 2014, 21, 4-6.

/2/ T. Skálová et al., Mouse Clr-g, a Ligand for NK Cell Activation Receptor NKR-P1F. Crystal Structure and Biophysical Properties, J. of Immunology, 2012, 189, 4881-4889.

This work was supported by the project P302/11/0855 of the Czech Science Foundation, BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, LG14009, and MSMT EE-2.3.00/30.0029BIOPOL.