Protein crystallization is a key step in enabling structural studies using crystallographic methods. Despite much research and experimental progress in this area, there is still no clear model to explain the mechanisms and physicochemical principles for all of the stages of crystallization. Studying the mechanisms of crystallization at the earliest possible stage of the procedure, including rapid characterization of the solution and protein solubility, would greatly enhance the prediction of potential crystal formation.

The process of the protein cluster formation was discussed and the attempts to observe it were made, but the structure of the formed clusters remained unknown. Recently, we have proposed the hypothesis that there is a pre-crystallization phase in solution composed of protein oligomers, where these oligomers are the elements of the resulting crystal structure. On the base of crystal structure of tetragonal lysozyme crystals octomer cluster was selected as a possible element of crystal growth [1]. These octamers were found in crystallization solutions by small-angle X-ray and neutron scattering methods (SAXS and SANS). The results show noticeable presence of lysozyme dimers and octamers under crystal growth conditions and total absense of oligomers under conditions when crystal growth is impossible. The influence of the precipitant cation type in a series of chlorides (NaCl, KCl, LiCl, NiCl2, CuCl₂, CoCl₂) on the structure of lysozyme solutions was also investigated [2]. The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of these metals are analysed on the basis of crystal structure data [3].

The relationship between various parameters of crystallization solutions and the possibility of protein oligomers formation under selected conditions could be useful for ordered films formation. The formation of the Langmuir films from crystallization solutions were studied by grazing-incidence X-ray standing waves [4]. Studying the formation of these monolayers by GIXSW showed that the thickness of the resulting protein layer was twice the diameter of an individual lysozyme molecule and matched the diameter of the octamer. Thin layers of precipitant ions (K and Cl) formed directly under the protein monolayer.

[1] Kovalchuk M. V., Blagov A.E., Dyakova Y.A., Gruzinov A.Y., Marchenkova M.A., Peters G.S., Pisarevsky Y. V., Timofeev V.I., Volkov V. V. (2016). Crystal Growth & Design 16 (4), 1792.
[2] Dyakova Y.A., Boikova A.S., Ilina K.B., Konarev P. V., Marchenkova M.A., Pisarevsky Y. V., Timofeev V.I., Kovalchuk M. V. (2019). Crystallography Reports 64 (1), 11.
[3] Marchenkova M.A., Kuranova I.P., Timofeev V.I., Boikova A.S., Dorovatovskii P. V., Dyakova Y.A., Ilina K.B., Pisarevskiy Y. V., Kovalchuk M. V. (2019). Journal of Biomolecular Structure and Dynamics in press, 1.
[4] Kovalchuk M. V., Boikova A.S., Dyakova Y.A., Ilina K.B., Konarev P. V., Marchenkova M.A., Pisarevskiy Y. V., Prosekov P.A., Rogachev A. V., Seregin A.Y. (2019). Thin Solid Films 677 (February), 13.

This study was supported in part by the Ministry of Science and Higher Education within the State assignment FSRC «Crystallography and Photonics» RAS and by the Russian Foundation for Basic Research (project number 18-32-20070 mol_a_ved).

The formation of structured oligomers in protein solutions during crystallization by SAXS and SANS methods are shown. The experimental SAXS and SANS data are processed using models of oligomers extracted from the crystal structure. Octamers and dimers are formed in a crystallization solution during growth of tetragonal lysozyme crystals with the addition of precipitant NaCl [1]. The volume fraction of octamers increases with the protein concentration increase and the temperature decrease. Addition of (NH₄)₂SO₄ or NaNO₃ as precipitants is shown to induce the formation of a significant fraction of protein dimers in proteinase K solution during the growth of tetragonal crystals [2]. Hexamers are formed in crystallization solution during hexagonal crystal growth of thermolysin [3]. The hexameric volume fraction increases when the supersaturation conditions are met, i.e. when the temperature decreases and the precipitant ((NH₄)₂SO₄) concentration increases. The formation of transaminase dodecamers is shown in crystallization solution with addition of precipitant NaCl [4]. Protein crystal and its oligomers are presented in the figure 1-a. Oligomers may act as building blocks in the growth of proteins single crystals. Also, the influence of solvent type (Н₂О and D₂O) on structure crystallization solution was investigated [5]. The dimer and octamer formation in crystallization solution in Н₂О and D₂O is shown.

The volume fraction of octamers increases with a decrease in temperature in both type of solvent (figure 1-b). Concentration of octamer is higher in crystallization solution in D₂O then in H₂O.

[1] Boikova, A.S., Dyakova, Y.A., Ilina, K.B. et all. (2017) Acta Crystallographica Section D: Structural Biology, 73 (7), 591
[2] Boikova, A.S., D’yakova, Y.A., Il’ina, K.B. et all.Crystallography Reports, 63 (6), 865
[3] Kovalchuk, M.V., Boikova, A.S., Dyakova, Yu.A. et all. (2019) Journal of Biomolecular Structure and Dynamics, 37 (12), 3058
[4] Marchenkova, M.A., Konarev, P.V., Rakitina, T.V. et all. (2019) Journal of Biomolecular Structure and Dynamics, in press
[5] Boikova, A.S., D’yakova, Y.A., Il’ina, K.B. et all. (2017) Crystallography Reports, 62 (6), 837

This study was supported in part by the Ministry of Science and Higher Education within the State assignment FSRC «Crystallography and Photonics» RAS and by the Russian Foundation for Basic Research (project number 18-32-20070 mol_a_ved).

Using the molecular dynamics simulation method, the stability of lysozyme octamer and two types of dimer (A and B) formed in solution under conditions of crystallization of tetragonal syngony was studied. In order to investigate the influence of NaCl precipitant ions bound to the protein in the crystal, various combinations of sodium and chloride ions associated with lysozyme molecule were probed: 1) with Na and Cl ions, 2) only with Na ions, and 3) without any ions. Using the GROMACS program, 100-ns molecular dynamics trajectories of the oligomers in the presence and absence of precipitant in water were calculated at different temperatures from 278 to 318 K.

To evaluate the stability of oligomers, RMSF (Root Mean Square Fluctuations) graphs were plotted at every simulated temperature.

As a result, flexibilities of octamer and dimer A have regularly increased with the temperature growth only in the case of considering precipitant ions embedded in the crystal structure. The RMSF values of dimer B are approximately the same at temperatures from 283 to 313 K and become higher at 318 K for all simulations whether they were performed with bound precipitant ions or not.

Thus, the importance of Na and Cl ions associated with the lysozyme is shown as only results of simulating oligomer models containing precipitant ions are consistent with the ones obtained by small-angle x-ray scattering experiments on crystallization lysozyme solutions [1-2].

[1] Kovalchuk, M. V., Blagov, A.E., Dyakova, Y.A., Gruzinov, A.Y., Marchenkova, M.A., Peters, G.S., Pisarevsky, Y. V., Timofeev, V.I., Volkov, V. V. (2016). Crystal Growth & Design 16 (4), 1792.
[2] Boikova, A. S., Dyakova, Y. A., Ilina, K. B., Konarev, P. V., Kryukova, A. E., Kuklin, A. I., Marchenkova, M. A., Nabatov, B. V., Blagov, A. E., Pisarevsky, Y. V. & Kovalchuk, M. V. (2017). Acta Cryst. D73, pp. 591-599

Poly(N-isopropylacrylamide) (PNIPAM) is a representative thermoresponsive polymer, and its aqueous solution becomes phase separated at a temperature higher than the cloud point (T_cp). To further promote the use of PNIPAM as a functional material, its phase behavior should be understood as a first benchmark. Previously, it was pointed out that the viscosity of a PNIPAM aqueous solution with a relatively higher concentration dramatically increased with temperature near to the T_cp. Although the origin of the peculiar viscosity increase just below the T_cp is a key to understanding the phase behavior, it remains unclear at the moment. Thus, it is necessary to examine the structure and physical properties of the PNIPAM solution a temperature just below the T_cp on various length scales. In this study, we report on the structure and physical properties examined by small-angle X-ray scattering (SAXS) measurement in conjunction with a particle tracking, in which the thermal motion of probe particles in the solution was tracked.

An aqueous solution with a concentration of 10 wt% was prepared from atactic PNIPAM with a number-average molecular weight of 193k and a polydispersity index of 2.2. The T_cp of the PNIPAM solution was determined by monitoring the light transmittance at a wavelength of 633 nm passing through the solution, as a function of temperature. A temperature (T_cp’), at which the transmittance started to decrease upon heating, was collected at different heating rates (n). By extrapolating the T_cp’ value to n = 0, the T_cp value was determined to be 304 K. SAXS experiments were performed at the BL03XU in SPring-8, Japan.

Fig. 1(a) shows photographic images of the time-variation in the physical state of the PNIPAM-water mixtures. When the PNIPAM-water mixture was aged for 3 h at 302 K, which was just below the T_cp, it turned into a transparent gel without any loss of transparency. Such a gelation was not observed at 294 K. Fig. 1(b) shows SAXS profiles for the mixtures that were aged for 3 h at 302 and 294 K. The contribution of water to the scattering for the sample was subtracted. In general, a scattering from the polymer chains in semi-dilute solution, where the polymer concentration is above the overlap concentration, can be described by the Ornstein−Zernike (OZ) function. In fact, the experimental data obtained for the mixture aged at 294 K could be reproduced by the OZ function, as drawn by solid lines in the panel (b). The value of x, which corresponded to the average size of blobs consisting of polymer segments, was 2.6 nm. On the other hand, the data for 302 K could not be fitted by the OZ function. The scattering intensity in the range smaller than 0.6 nm⁻¹ was intensified. To express the data in the small q region, the scattering function based on the Guinier model, which was used for an aqueous solution of isotactic PNIPAM, was included as an additional term [1]. The x, and R_g values obtained by the curve fitting were 5.9 and 7.5 nm, respectively. According to the previous result, the R_g value should correspond to the size of the clusters formed by intermolecular association of segments, as shown in Fig. 1(c). In the presentation, the results of the particle tracking will be also given to discuss a possible mechanism for the gelation [2].

The phase separation relationship between polymer and fullerene have been treated as a major influence that impact the operation of bulk heterojunction solar cells. In this research, we investigate the intercalation behavior between PBTTT-C14(PBTTT (Poly[2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene])) and PCBM ([6,6]-Phenyl C71 butyric acid methyl ester, PC71BM) to unveil the phase separation relationship in this system.

Temperature dominates the competition between the intercalation of PCBM into PBTTT-C14 order phase and self-crystallization of PCBM. At lower temperature, PCBM tends to intercalate into the cavity among PBTTT-C14 side chain. However, at higher temperature PCBM tends to crystallization.

In the other hand, PCBM are not able to intercalate into PBTTT-C14 domain once the side chain regularity is reduced by partially soluble solvent on PBTTT-C14. No self-assembling relationship are found between PBTTT-C14 and PCBM either at high or low temperature.