Introduction
Protein crystallization has been used for decades for structure determination of detailed molecular structure of proteins in atomic resolution. Millions of successfully performed crystallization experiments by the trial and error method have shown that the presence of other substances is practically always necessary for the regular ordering of protein molecules into the crystal. The very laborious development of crystallization screens has led to the massive deployment of large crystallization robots that control the precise setting of the composition and the change of the chemical parameters. Currently, the successful crystallization is expected in about one quarter of new proteins.
However, the fundamental problem is that almost everything is based on practical experience and there is no trustful and logical explanation of the principle on which crystallization agens work. Neither is clear when to use which crystallization agens nor whether it is appropriate to combine them with each other.
Dynamic Theory of Protein Crystallization
Our Dynamic Theory of Protein Crystallization (DTPC) hopefully explains all the uncountable crystallization experiments that have been described during the last decades of experience with protein crystallization [1].
A practice of the last decades clearly showed that the successful protein crystallization requires a presence of additives directly influencing regular stacking of protein molecules in the crystal. We call them Protein Crystallization Agens (PCA). By definition these special crystallization additives control amounts of miss-placed and miss-oriented molecules in the growing solid. Thus, they practically control the difference between the crystalline and amorphous solid state. Do not misinterpret them with another crystallization additives added for different reasons. Imagine also that some chemicals can play several important roles in the crystallization solution.
Another important point is that the protein in the oversaturated crystalline solution are highly influenced by the specially added PCA. It is well proved that in the molecularly overcrowded crystallization solution, there is high concentration of temporary protein-PCA adducts (P-PCA adducts). Properties of the adhesion patches on protein surface and also the orientation of the P-PCA adduct with respect to its motion in solution will change significantly in comparison with the naked protein. Non-covalent bonds between protein and PCA should be weak to allow fluent release of PCA molecules from the growing crystal back to solution.
It is obvious that very large surface of protein molecules always has many adhesion patches that compete with each other when depositing the protein molecule into the crystal. It is also evident, that if protein join to the crystal surface in an incorrect way, it will definitely stay as a stacking fault. It is improbable, that the molecule will dissolve again, turns to the correct orientation and join the surface again. Thus, the classical thermodynamics is not sufficient. The physical laws of rigid body motion in liquids play an important role here.
This presentation will show some examples of how the experimenter influences the way in which protein molecules stack in the growing crystal and also how the crystal seeds grow from the beginnings.
We will show the function on examples of experimentally best confirmed crystallization agens :
In the Appendix, there is a list of definitions useful in the Dynamic Theory of Protein Crystallization (DTPC).
The work was supported by the Czech Science Foundation 25-17546S.
[1] Hašek, J., (2011) Principle of the unique adhesion mode in protein crystallization, Acta Cryst. A67, C537;
[2] McPherson, A. (2001) Comparison of salts for the crystallization of macromolecules, Protein Science, 10418-10422.
[3] Kimber,M.S. et al, (2003) Crystal screen optimization. Data mining crystallization databases: Knowledge-based approaches to optimize protein crystal screens, Proteins, 51, 562-568.
Appendix – Hašek J. - Protein Crystallization - Structure 2025
Definition of terms – Glossary
Glossary summarizing some terms important for
the dynamic theory of protein crystallization and their abbreviations used in this work
Aditives Different compounds utilized for preparation of crystallization solution called additives. Some additives have more functions in the crystallization solution. Their reported purposes are:
· to control stacking the protein molecules in the growing crystal
· to increase solubility of the protein, prevent its aggregation,
· to control nucleation,
· to modulate crystal habit,
· to optimize buffer conditions,
· to modulate pH and ionic strength,
· to stabilize the protein,
· to protect denaturation
· to protect formation of ice during flesh freezing
· to control of viscosity of solution to slow down flows and diffusion, etc.
PCA Protein crystallization agens directly influencing the stacking of protein molecules in the growing crystalline phase
VA Viscosity agens - added to increase viscosity of the solution to slow diffusion
Protein-PCA aduct Protein molecule temporarily linked to (non-covalently clustered) the molecules of protein crystallization agens (PCA)
Precipitant - Precipitation agens – the compound with high affinity to water molecules reducing thus amount of free water molecules in solution
CTPC Classical theory of protein crystallization considers crystal growth as a regular sedimentation of ideally dissolved (uncomplexed) protein molecules into the crystalline phase in some pre-defined environment. A regular stacking of protein molecules is derived from thermodynamic parameters.
DTPC Dynamic theory of protein crystallization takes into account formation of temporary molecular aggregates in the crystallization solution. The activities of the temporary aggregates are important particularly during the crystal initiation phase. It recognizes the non-equilibrium nature of protein crystallization and analyses the kinetics of intermediate states during crystallization process and explains why the correct setting of the crystallization experiment leads to regular crystals instead of irregular sediment during the solidification process..
Direct observations of the temporary nano-scaled molecular processes in the highly concentrated solutions are usually difficult. The proof of intermediate processes is validated usually by indirect macroscopic observations.
AP Adhesive patch is the region on the surface of a protein molecule responsible for adhesion to the adjacent molecule. Protein have usually many adhesive patches on its surface – but, only some of them can form intermolecular contacts in a given configuration of molecules in the solid state.
PPAM Protein-protein adhesion mode. It is an abstract term describing a tendency to some specific adhesion between two or more protein molecules. It does not describe an exact geometry of the molecular adduct. The particular spatial realization of the contacts depends always on the molecular environment (the composition of the crystallization solution, the phase, etc.). It means that the complementary patches on surfaces of the adhering proteins differ in different environments. Despite these differences, it is usually easy to identify any particular adhesion mode in different molecular environments such as dilute solution, concentrated solution, amorphous, crystalline phase, in living tissue, etc.
DAM Dominant adhesion mode is the PPAM leading to the highest decrease of free energy of system in a given environment. It depends on the molecular environment and thus can be changed by experimenter, e.g. by composition of crystallization solution. Thus, DAM is not a property of a given protein compound only. It can be changed by experimenter.
PDAM Principle of the Dominant Adhesion Mode. It is the evident principle saying that only one PPAM must dominate crystallization to get a regular crystalline phase. It must be respected by any crystallization method.
SAM Subsidiary adhesion modes are the PPAM compatible with the dominant DAM in a given space group. The free energies of the DAM and SAMs are decisive for the crystallization rates in the respective crystal directions.
IAM Incompatible adhesion mode. The protein-protein adhesion mode (PPAM) which is incompatible (cannot coexist) with the dominant adhesion mode (DAM) in a given crystalline form.
MPPI Modulators of Protein-Protein Interaction. The compounds temporarily modifying the adhesion properties between the target protein molecules. There are several important subclasses of these compounds described bellow.
PSAM Protein Surface Active Molecules. The molecules forming temporary adducts with the target protein molecules and changing thus the adhesive properties of the protein-PSAM adduct envelope with respect to the envelope of the naked protein. Temporary covering the specific patches on the protein surface influences the stacking of protein molecules into the growing crystal. Many efficient PSAMs are already used in crystallization experiments as precipitants or as additives.
PSSA Protein Surface Shielding Agens the molecules designed to bind the protein surface patches important for crystallization in an unwanted crystal form.
PPLM Protein-Protein Linking Molecules are the molecules designed to bind surfaces of two protein molecules forming thus a temporary molecular adduct in solution. The PPLM molecules are sometimes observed in structures deposited in the PDB
CSAM Compatible Set of Adhesion Modes. The set of cooperative adhesion modes which are mutually compatible within a given crystal form.
PXAM Protein-PSAM Adhesion Mode. Due to the large variability of protein surface, small molecules have usually many adhesion patches on the protein surface.
TPA Temporary protein adducts are semi-stable complexes of the protein molecules with some other molecules. If the adducts are stable enough, the adjoining molecules can effectively block an access to some areas on the surface of the target protein molecule.
Initiation of protein crystallization
CI Crystallization initiator (catalyst) is a heterogeneous object inducing protein crystallization. It acts as a catalyst for the formation of stable and regular crystal nuclei. Stable nuclei can separate and continue to grow far from the surface of the CI.
PIC Porous initiator of crystallization is the CI, where its morphology has dominant effect on crystallization.
CN Crystallization nucleus (crystal seed) is a crystalline aggregate of molecules that has potential to grow under suitable conditions into a stable crystal.
Substrate is material used to prepare the CI. It need not show any nucleation properties itself.
Nucleant is a particle or some specific surface element that triggers the nucleation of the target material.
MIP Molecularly imprinted polymer – polymer with pre-formated cavities imprinted by some molecular objects.
Cognate MIP The MIP imprinted by the target protein molecule.
NIP Corresponding Not Imprinted Polymer as a negative evidence in the MIP activity tests.
CNM Carbon Nano-Material is characterized by a large ratio surface to volume -– for example carbon nanotubes, or carbon “fractal” nanoparticles (e.g. carbon black).
Ordering in solution
Kosmotropic agens – the molecules or ions supporting ordering of the neighbor solvent molecules and stabilizing thus the protein structure. They support formation of well-ordered 3D sets of intermolecular interactions.
Chaotropic agens – the molecules or ions exerting chaotropic effect in solution and generally destabilizing protein conformation. Their presence brakes formation of a regular net of hydrogen bonds between water molecules in solution.
Malonates the compounds forming malonate anion in solution ( -OOC-CH2-COO- )
Polymers
Polyether Class of polymers with the repeating pattern R-O-R’. Smile notation OROROR…ORO
Crown ether Cyclic oligomers of polyether with outstanding and tunable chelating ability to cations and to the hydrogen-bond-donor molecules.
PEG = Poly(ethyleneglycol) = poly(oxyethylene) = poly(ethyleneoxide) = macrogol.
Polymers with chemical formula H−(O−CH2−CH2)n−OH (Smile notation OCCOCCOCC…OCCO).
High flexibility of the PEG chain allows formation of loops where the loan electron pairs of successive ether oxygens orient to the a single center binding thus selectively some cations. They act also as eficient hydrogen-bond-acceptors forming relatively stable adducts with hydrogen bond donor. PEG belongs also to the class of polyethers.
Database of protein-polymer interactions (DPPI) – set of protein structures selected from the PDB (Protein Data Bank), containing polymer fragments. It involves also the imaging tools allowing easy viewing and the serial analysis of intermolecular contacts of individual polymer fragments to the protein surface. It greatly simplifies analysis of the protein-PSAM adhesion modes.
LB films Langmuir-Blodget films. Well-ordered films composed of monomolecular layers of macromolecules used by Pechcova and Nicollini for the preparation of protein crystal initiators.
PDMS PolyDiMethylSiloxane. The polymer used by Ghatak et al for preparation of roughly waved surface by an incision of the stretched folies.
NHMA N-hydroxymethylacrylamide. The polymer used as a matrix for molecular imprints by Saridakis et al.