Intermolecular interactions displayed by X-ray diffraction

J. Hašek¹, J. Dušková¹, T. Skálová¹, T. Koval² and J. Dohnálek^1,2

¹Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovského nám. 2, 162 06 Praha 6, Czech Republic

²Institute of Physics, Academy of Sciences of the Czech Republic, v.v.i., Na Slovance 2,182 21 Praha 6, Czech Republic,

hasek@imc.cas.cz

X-ray diffraction methods have been developed in a user-friendly and relatively quick method for determination of macromolecular structure, for analysis of molecular dynamics and for determination of reaction paths taking place in these systems. In principle, the method has not any limitations as far as the complexity of the bio-macromolecular adducts. The results are fully based on experiment (no a priory assumptions on structure neither molecular modeling are required) and thus they are very reliable in comparison with any other method. Accuracy of atom positions depends always on quality of the local electron density, but for typical data measured at 1.8 Å resolution the expected accuracy is about 0.1 Å.

Protein crystal can be defined as a regular three-dimensional scaffold of protein molecules stabilized in solution by weak intermolecular forces. “The crystals” are in hydrodynamic equilibrium with solvent and must be measured in solution. Insensitive changes in solvent composition or solvent contents in the crystal quickly destroy diffraction quality of the sample.

Protein crystals can exist in various concentrations (protein contents from 15 % to 75 %). However, for each molecular stacking, there is only a narrow range of solvent contents and the ingredient concentrations under which the stacking preserves its periodicity.

X-ray structure analysis is a very reliable method and thus if one observes adhesion between macromolecules, it is a definite prove that this interaction exists under conditions present in the measured sample. However, because of large surface of macromolecules, each protein has several different interaction modes with preferences dependent on the solvent composition. Thus, the diffraction methods can show as a rule several complexes between two proteins with different affinity and stability. All the observed adhesion modes are real under given circumstances. The decision which adhesion mode dominates in the biological process in the cell may be simple or complex, but the preparation of several crystal forms showing all adhesion modes is desirable to get full view of the bio-processes in any case.

The real adhesion mode dominating in the bioprocess under investigation can be different from the adhesion modes observed in solution because of many competitive intermolecular interactions and spatial restrictions in the living cell. Even more, the biologically relevant adhesion mode need not be the most stable in solution, because the proteins are often oriented by anchoring in membrane or are simply temporary complexed with other molecules forming the respective organelle of the cell. Thus, many unknown factors can change the adhesion properties of the inspected macromolecule in the bioprocess under investigation.

X-ray structure analysis is optimal tool for inspection of these phenomena, because the crystalline samples studied have very similar concentration of the protein material as it is in organelles of the living cell (see Figure 1). The talk explains how “protein surface modifying agents PSMA” [1, 2] allow growing different crystal forms showing different adhesion modes between proteins to get as far as possible a complete review of all adhesion modes of the protein studied. It also explains a role of PSMA in increasing the accuracy of the experimentally determined atom positions under the desirable limit 0.1 Å (see Figure 2). Applications of polymer materials as “protein surface modifying agents” and behaviour of hydrophilic polymers in the bio-environment are summarized in [3]. The practical usage of these considerations in development of new crystallization screens were published in [4]. It is expected, that the new more complex usage of the X-ray diffraction methods as described here can provide more realistic insight on the real behaviour of molecules in their natural bio-environment.

Figure 1. The graph shows a water contents in bio-macromolecular structures deposited in the PDB until 2012. It shows that a regular stacking of macromolecules into protein crystal is not possible when protein concentration in “the solid phase” is lower than 30 % or higher than 70 %. To compare it with a standard biological system: the dark rectangular area shows an average concentration of all biomolecules in cells and the lightly coloured area shows their expected concentrations in cell organelles.

Figure 2. A higher content of water in the measured protein crystal leads on average to numerically higher “resolution” (to lower amount of diffraction data). The “protein surface modifying agents PSMA” can help to stabilize the macromolecular stacking in regular lattice and to get the resolution under the desirable limit 1.8 Å corresponding roughly to the accuracy of atom positions about 0.1 Å on average [3, 4].

The work on this project was supported by the Grant Agency of the Czech Republic, project no. 305/07/1073, P302/11/0855 and Grant Agency of the Czech Academy of Sciences IAA500500701.

1. Hašek, J. Zeitschrift fur Kristallografie, 2006, 23, 613-618.   

2. Hašek, J. Journal of Synchrotron Radiation, 2011, 18, 50-52.

3. Hašek, J. , Skálová, T., Dušková, J., Kolenko, P., Koval, T., Dohnálek, J. Crystallography Reviews, 2012, in press.

4. Skálová, T., Dušková, J., Hašek, J., Kolenko, P., Štěpánková, A, Dohnálek, J. J. Appl.Crystallogr., 2010, 43, 737-742.