Investigation of the Initial Crystallization Stage in Lysozyme Solutions

Subscriber access provided by LAURENTIAN UNIV

Communication

Investigation of the initial crystallization stage in lysozyme solutions by small-angle X-ray scattering Mikhail V Kovalchuk, Alexander E. Blagov, Yulia A. Dyakova, Andrey Yu. Gruzinov, Margarita A. Marchenkova, Georgy S. Peters, Yury V. Pisarevsky, Vladimir I. Timofeev, and Vladimir V. Volkov Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.5b01662 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 26, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

Investigation of the initial crystallization stage in lysozyme solutions by small-angle X-ray scattering Mikhail V. Kovalchuk a,b,c, Alexander E. Blagovb,a, Yulia A. Dyakovab,a, Andrey Yu. Gruzinova, Margarita A. Marchenkova*,b,a, Georgy S. Petersa, Yury V. Pisarevskyb,a, Vladimir I. Timofeevb,a and Vladimir V. Volkovb,a a

b

National Research Centre “Kurchatov Institute”, 123182, Moscow, Russian Federation Shubnikov Institute of Crystallography of Russian Academy of Sciences, 119333 Moscow,

Russian Federation c

St. Petersburg State University, 199034, St. Petersburg, Russian Federation

KEYWORDS. Crystal growth unit, protein solution, small angle X-ray scattering, molecular modelling, lysozyme.

ABSTRACT: From the base structure of tetragonal lysozyme crystals, the octamer cluster was selected as the possible element of crystal growth. The proposed model was verified by smallangle X-ray scattering (SAXS) measurements of lysozyme solutions. The results showed a noticeable presence of lysozyme dimers and octamers under crystallization conditions and the total absence of oligomers under conditions where crystal growth was impossible.

Introduction

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 19

The crystallization of proteins is an extensive area of research, but a very important and difficult challenge in this area is to find the proper crystallization conditions. At the present time, a number of methods for protein crystallization have been developed

1, 2

. However, the

crystallization conditions are still too often governed by trial-and-error methods; hence, the ability to obtain protein crystals with high X-ray diffraction qualities is not very predictable 3. The quality of grown crystals depends on many parameters, e.g., the method of growth, rate of evaporation, concentration of precipitant and protein, and the temperature 4, as well as the nature of the substance transport to the growing crystal

5-8

. Currently, the commonly used protein

crystal growth methods are based on a statistical analysis of growth experiments conducted over a wide range of crystallization conditions 9. Protein crystals are used for obtaining the protein structure. The study of crystallization mechanisms is important not only to build a conscious scheme for the selection of protein crystallization conditions to determine the structure of molecules but also to find ways to create hybrid systems that include protein molecules as functional elements. A detailed study of the mechanism and processes that occur at the beginning of nucleation and during protein crystal growth will facilitate the development of methods for predicting crystallization conditions and for controlling the crystallization process. One of the most important aspects is to define the structure of the basic physical units that are connected step-by-step to a growing nucleus in building the entire crystal. The possibility of forming complexes in a supersaturated solution has been discussed in some studies on the process of protein crystallization 10-16. Among the works devoted to elucidating the crystallization process of macromolecular compounds, the most widely studied process is the crystal growth of the tetragonal form of


2

Page 3 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


lysozyme from a chicken egg (HEWL) because of the ease of crystallization and the availability of the reagent. A substantial amount of information on the crystallization conditions of the tetragonal form lysozyme is available in the literature. Ducruix et al. 17 used small-angle X-ray scattering (SAXS) experiments to demonstrate that the interactions between particles were modified from repulsive to attractive by the addition of a precipitant. Moreover, a temperature and differential anion effect was observed, which affected the solubility and interactions in HEWL solutions 18. In particular, Pusey et al.

19

suggested that oligomers in the protein with the precipitant

solution (dimers and tetramers) were involved in the growth of the crystal. Such oligomer models have been used to explain atomic force microscopy results related to the growth rates of the faces 20. Boué et al.

21

investigated the crystallization solution of HEWL by small-angle neutron

scattering (SANS). They showed that under the crystallization conditions, the radius of gyration was larger than that in a monodisperse system of monomers but lower than that in a monodisperse system of octamers. They also demonstrated that the radius of gyration increased with decreasing solution temperature. Unfortunately, the angular dependences of neutron scattering were investigated for only a small angular range. These results did not provide conclusive evidence concerning the types and size distributions of oligomers in the solutions. The authors did not obtain reliable quantitative reproducibility, possibly because of their sample preparation technique (whether filtration and centrifugation procedures were performed with the protein solution is unclear). In all of these previous studies, not only monomers but also aggregates of a few molecules were assumed to be involved in the growth of the crystal. The nature and structure of these


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 19

protein aggregates have not been determined, and they have not been assumed to be ordered. Moreover, definitive experimental evidence on the correlation of complex formation in the solution and the initial step of the crystallization process has not been obtained. Finally, the process of protein crystal growth at the molecular level has not been determined and the structure of the oligomers directly involved in the crystal growth remains obscure. The objective of our work is to study the crystallization mechanisms, identify the structures of possible crystal growth units, and study the relationship between crystal formation in the solution of certain oligomer types and the optimum crystallization conditions. We hypothesized that a cluster from the crystal structure can act as a growth unit to provide crystal growth. In this paper, we selected elements in the crystal structure of tetragonal lysozyme crystals, which are ordered oligomers, and assumed that these oligomers can function as growth units in crystal growth. Experimental confirmations of our assumptions were obtained by investigating the presence of complexes in solution, which provided growth of the tetragonal lysozyme crystals. According to Heijna et al.

22

, several types of lysozyme crystal structures could be grown: tetragonal,

monoclinic, orthorhombic, and triclinic. We studied the growth of the tetragonal form of lysozyme because it was formed in the widest range of conditions and was the most stable. We investigated the various states of protein molecules in the crystallization solution by SAXS using high-intensity synchrotron radiation. SAXS is one of the most effective methods of experimental research on organic nanoscale structures in solution 23. This method allowed us to study the crystallization process at the earliest stages. In addition, the use of a high-intensity synchrotron source (NRC “Kurchatov Institute”) allowed us to use shorter exposure times to prevent the destruction of the sample.


4

Page 5 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In this work, comparing the structure of the protein solution under conditions when crystallization was impossible with the structure under optimum conditions for producing high quality tetragonal lysozyme crystals was important. Saturation of lysozyme could be controlled by the protein concentration, buffer ionic composition, or the temperature. We here studied the process of crystal growth as a function of temperature at a constant concentration of protein and precipitant. The strong temperature-dependent solubility of lysozyme allowed for the measurement of oligomer concentrations in both saturated and unsaturated solutions 24. Molecular modelling based on the structural analysis of tetragonal lysozyme The crystal structure of lysozyme from the Protein Data Bank (PDB ID: 4WLD) determined with a resolution of 1.54 Å was chosen to construct the molecular models of possible growth units of the tetragonal lysozyme crystals. The crystal belongs to the P43212 space group with unit-cell parameters of a = b = 79.20 Å, c = 37.90 Å, and α = β = γ = 90.00°. The solvent content in the crystal was 40.68%, and the asymmetric unit contained one lysozyme molecule. Using the program Coot

25

, we propagated the asymmetric unit in space using symmetry operators

representative of space group P43212 (-X,-Y,1/2+Z; 1/2-Y,1/2+X,3/4+Z; 1/2+Y,1/2-X,1/4+Z; 1/2-X,1/2+Y,3/4-Z; 1/2+X,1/2-Y,1/4-Z; Y,X,-Z, -Y,-X,1/2-Z). Determination of the growth units on the basis of the structural analysis of tetragonal lysozyme Figure 1 (centre) shows a projection of the tetragonal lysozyme structure perpendicular to the 4-fold axis. The analysis of the crystalline molecular packing of lysozyme shows that the motif is repeated; this motif can be assembled by translating the complete octamer crystal structure and can be identified in three main ways. Next, using the program Coot and PyMOL 26, we obtained the coordinates of two octamers. The clusters (the growth units), as evident in Figure 1 (A–left


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 19

and B–right), differ in configuration and volume (size). One of them is formed by the multiplication of the asymmetric unit by the 4-fold screw axis, and the other is formed by combining two tetramers with reproduction of the asymmetric unit along the 2-fold simple and screw axes of symmetry. The crystal packing for hexamers, tetramers, and dimers of the lysozyme molecules was obtained from the octamers by successive removal of the lysozyme monomers. Each of the resulting clusters was saved as a file containing the atomic coordinates. The models of the two selected octamers and the other oligomers, which are their components, were used in the processing of the experimental SAXS curves.

Figure 1. A projection of the tetragonal HEWL structure (PDB ID: 4WLD) perpendicular to the 4-fold axis (centre). Two octamers (A and B) are proposed as growth ‘building blocks’. Lysozyme molecules are marked with different colours for clarity. Two different octamers (A–left and B–right) from the lysozyme structure are proposed as growth ‘building blocks’. The protein molecules are marked with different colours for clarity. The volumes of octamer A and octamer B are 117 nm3 and 140 nm3, respectively. Experimental Section


6

Page 7 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Materials and sample preparation Lysozyme from a chicken egg (HEWL) (CAS# 12650-88-3) was purchased from SigmaAldrich; sodium acetate (CAS# 6131-90-4) and NaCl (CAS# 7647-14-5) were purchased from Helicon. The crystallization conditions were previously chosen at the IC RAS laboratory 27. The protein was dissolved in 0.1 M sodium acetate buffer (SAB). After the solution was filtered through a 0.22 µm Millex membrane filter, it was centrifuged at 10,000 rpm for 10 min to remove protein aggregates and large particles. The final protein concentration (80 mg/ml) was adjusted while being monitored by absorbance measurements at 280 nm in 1.0 cm cells with a Cary 5000 spectrophotometer (Varian, Australia). NaCl, dissolved to a final concentration of 50 mg/ml in the same buffer, was used as the precipitant. All solutions were prepared in ultrapure water (Milli-Q, Millipore, 18 MΩ·cm). A solution of protein mixed with precipitant (1/1, v/v), protein (40 mg/ml), NaCl (25 mg/ml) and buffer solutions were investigated by the SAXS method. SAXS data from the buffer were subtracted after all measurements were calibrated to zero absorption. The study of lysozyme conformation was conducted under different temperatures in the range from 10 to 40°С. SAXS measurements SAXS experiments were performed on the DICSY synchrotron beamline (NRC Kurchatov Institute, Moscow)

28

at a constant wavelength of 1.62 Å. SAXS patterns were obtained using a

Pilatus3 1M detector with a sample-to-detector distance of 300 mm and an exposure time of 5 min. The primary beam was collimated using a three-slit scheme, which provided a crosssectional beam size of approximately 0.4 × 0.6 mm2 on the sample. The angular scale was refined using a silver behenate sample

29

. Initial data reduction was performed using the Fit2D

package 30 and Primus (from the ATSAS package) 31.


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 19

Samples were measured in thin-walled (0.01 mm) glass capillary tubes with diameters of 1.5 and 2 mm. The capillaries were placed into a brass sample holder connected to a watercirculating bath. The temperature was controlled with a digital sensor with a resolution of ±0.1°C placed near the sample. Each sample was exposed for 5 min prior to data acquisition. Test measurements with exposure times of 5, 10, and 20 min and long-term (30 min) data acquisition showed no significant differences in the scattering curves due to radiation effects and damage. The exposure time of the synchrotron studies was selected to avoid radiation damage of the samples. For this purpose, the primary beam cross section was fairly large and its intensity did not exceed 109 photons/s. SAXS data analysis Atomic models of oligomers were constructed from the tetragonal crystal structure of lysozyme (see Figure 1) and used as the input. Calculated scattering patterns from oligomers (including monomer) were used to fit the experimental data by a linear combination of components using the program OLIGOMER32. The quality of the fit is represented by the chi-square value χ2: 2

1 N  I mod ( s j ) − I exp (s j )  χ =  , ∑ N − 1 j =1  σ (s j )  2

(1)

where s = 4πsin(θ) / λ is the scattering vector, 2θ is the scattering angle, λ is the wavelength, σ(sj) is the standard error, N is the number of experimental points, Iexp(s) is the experimental dependence of the X-ray scattering intensity, and Imod(s) is the theoretical dependence of the Xray scattering intensity from the scattering vector, as calculated for the model mixture. Model SAXS curves were obtained from the atomic models using the CRYSOL program

33

.

To check for the presence of large aggregates, the scattering data were processed by the program MIXTURE 34 under the assumption of spherical shapes of macromolecules and their assemblies.


8

Page 9 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Such an assumption was justified by the fact that the shapes of the molecular complexes in this case were approximately spherical. Results and Discussion SAXS measurements of complexes in the lysozyme solution Lysozyme with no precipitation agent. SAXS curves were obtained for the protein solution with a concentration of 40 mg/ml at temperatures of 10, 25, and 35°C (Figure 2.) As evident in Figure 2, the scattering curves from the protein solution without a precipitant were similar to each other at different temperatures. The shapes of the curves revealed the influence of interparticle interference, which caused a relative decrease of the scattering intensity at low angles.

Figure 2. SAXS curves of a lysozyme solution (40 mg/ml) at different temperatures with no precipitant added. Black – 10°C; red – 25°C; blue – 35°C. Lysozyme with a precipitant. Figure 3 shows the results of the SAXS studies of a lysozyme (40 mg/ml) solution with a precipitant (25 mg/ml) in the temperature range from 10 to 35°C.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 19

Figure 3. SAXS curves of a lysozyme (40 mg/ml) solution with a precipitant (25 mg/ml) at different temperatures. Black – 10°C; red – 25°C; blue – 35°C. The addition of the precipitant caused a change in the interaction between molecules 17. In the case of a protein solution, when a precipitant was added at a concentration required to induce crystallization, the scattering curves differed substantially from each other at different temperatures. This result led to the conclusion that the distribution of molecules in the oligomer solution at different temperatures differs. This difference in distribution was also noticeable in the case of increasing temperature in the crystallization experiments, indicating a less aggregated state. Figure 4 shows the shape comparison of the SAXS curves for lysozyme solutions without (black) and with precipitant (25 mg/ml) (red) at 10°C. Adding the precipitant resulted in a decrease of interparticle interference due to changes in the state of protein molecules in the oversaturated solution. This effect is attributed to the formation of oligomers.


10

Page 11 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Shape comparison of SAXS curves for lysozyme solutions without (black) and with precipitant (25 mg/ml) (red) at 10°C. The volume fraction distribution of spherical particles according to their size and matching model of the scattering intensity is shown in Figure 5. The presented results were obtained using the MIXTURE software. The highest-intensity peak in the distribution corresponded to the monomer. Its half-width was determined by the molecule’s deviation from a spherical shape. The second, less-intense peak corresponds to units with an average diameter of 6.07 nm and a volume of 117 nm3, which is equal to the volume of octamer A (117 nm3). However, when the error is taken into account, we cannot completely exclude the presence of octamer B (140 nm3).


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 19

Figure 5. Volume size distribution of particles in a spherical approximation for the investigated samples: black – 10°C; red – 25°C; blue – 35°C. Peak 1 corresponds to the monomer, whereas peak 2 corresponds to octamer A. Therefore, to clarify the composition of the protein solution with a precipitant (in the crystallization conditions of the tetragonal form of lysozyme), we used the octamer A model and its corresponding dimer, tetramer, and hexamer models to fit the experimental data. Model SAXS curves for each component, as calculated using the CRYSOL software, are shown in Figure 6a. The data were interpolated by the least-squares method in the OLIGOMER software as a sum of the intensities of the model components, i.e., the atomic structures of monomers, dimers, tetramers, and octamers of lysozyme (Figure 6b). Despite exceeding unity, the shapes of the model curves were similar to the experimental patterns.

Figure 6. a) Model SAXS curves from lysozyme structures: Monomer (black), dimer (red), tetramer (blue), and octamer (green), as calculated by the CRYSOL software. b) Experimental scattering intensity (points) and model curves (lines) calculated from the composition of monomers, dimers, and octamers, as indicated in Table 1, by the OLIGOMER software: 1 – 10°C, 2 – 25°C, and 3 – 35°C. Curves are shifted vertically for better visualization.


12

Page 13 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1 presents the calculation results of the distributions of different oligomer types in the protein solution with the precipitant at 10, 25, and 35°C. At these temperatures, the lysozyme solubility in the presence of NaCl changed24. Therefore, the state of the protein and precipitant solution corresponded to three different regions in the lysozyme crystallization phase diagram 22. The first point (T = 10°C) corresponded to optimal growth, the second point (T = 25°C) corresponded to the region where the growth was hindered, and the third point (T = 35°C) corresponded to the area where the growth of tetragonal lysozyme crystals did not happen. Table 1. Relative concentration of single lysozyme molecules and its oligomers (A type) extracted from the SAXS curves at different temperatures: Lysozyme (40 mg/ml) with a precipitant (25 mg/ml).

Concentration, % Cluster type Т = 10оС Т = 25оС Т = 35оС Monomer

83±4

95±4

97.9±0.6

Dimer

8±4

1±4

0±0

Tetramer

0±0

0±0

0±0

Hexamer

0±0

0±0

0±0

Octamer

8.8±1.0

3.8±1.0

2.1±0.3

χ2

3.2

2.2

1.6


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 19

Because the solutions were tested at high protein concentrations similar to the conditions of crystallization, the shape of the scattering curves should be influenced by the effect of interparticle interference. Therefore, the amounts of aggregates found by our modelling should differ in their absolute values from the true quantities. However, in this work, we performed a relative comparison of the measurements at different temperatures and examined trends in changes of the composition rather than examining absolute composition. The effect of interparticle interference, which can complicate the treatment of experimental data, was also evident in the curves obtained from the lysozyme solutions without a precipitant at all temperatures (Figure 3). However, at this concentration, the average distance between the molecules (~ 17 nm) was approximately 6 times greater than the linear size of the molecules (~ 3 nm). In addition, when the precipitant was added, the molar concentration of the individual particles (monomers, dimers, and octamers) was reduced by approximately half compared to that in the protein solution without the precipitant. This result suggests that the changes in the interparticle interaction are within the experimental error. The use of lower concentrations in this case could lead to the avoidance of crystallization conditions and to a change in the composition of the mixture. A study of a series of concentrations to bring the SAXS intensity to a zero concentration was not applicable in this case. The SAXS curves for the selected models of oligomers matched the chi-square values χ2 from 1.6 to 3.2 (see Table 1). Large values of χ2 can be explained mostly by concentration effects and increased discrepancy at large angles. The numerical concentration of particles under crystallization conditions decreased because of their aggregation, which led to a reduction of the concentration effect (Fig. 4). An opposite influence of the shape of the scattering curves by the


14

Page 15 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


presence of aggregates and the structure factor led to oscillations of the scattering curves in the Guinier region. This effect was not observed in our measurements, which suggested a reduction of the impact of the structure factor. However, its influence led to increasing values of the chisquare tests in modelling the composition of the solutions by sets of aggregates. In our case, the systematic experimental errors due to nonmetering structure factors may lead to errors in the relative amount of aggregates to 20-30%. However, the main topic here was a comparative study of samples at different temperatures rather than the determination of the absolute concentrations of aggregates; thus, knowledge of the absolute errors was not required. Conclusion We assumed that the determination of growth units was possible on the basis of the protein crystal structure analysis. This assumption was confirmed by the solution SAXS results in the initial stage of lysozyme crystallization. The experimental SAXS data analysis based on two different models of octamers and their components showed that in the precrystallization state in solution, octamers and dimers were present, in addition to the monomers. Regardless of the chosen model of oligomers, tetramers and hexamers were not observed in the crystallization solution. As the temperature was increased and the protein solution moved from the crystallization region of the phase diagram, the amount of oligomers in solution decreased and became comparable to the error factors. In the presence of oligomers, the error factors increased, but their values were substantially smaller than the concentrations of dimers and octamers. The results from the crystallization model indicated a high probability that the crystal growth unit was combined from eight molecules of lysozyme. Hence, our approach could be useful for studying protein crystallization mechanisms. We identified the structures of possible crystal


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 19

growth units of tetragonal lysozyme and revealed the relationship between the formation of certain types of oligomers in solution and the optimum crystallization conditions. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This study was supported in part by the Russian Foundation for Basic Research (project no. 1302-12143_ofi_m, 15-29-01142_ofi_m, 16-32-60144_mol_a_dk). ACKNOWLEDGMENT The authors express their gratitude to V.R. Samygina, I.P. Kuranova, and B.V. Nabatov for consultation and discussion of the results. REFERENCES (1) Chayen, N. E.; Saridakis, E., Protein crystallization: from purified protein to diffractionquality crystal. Nat. Methods 2008, 5, 147-153. (2) Pechkova, E.; Gebhardt, R.; Riekel, C.; Nicolini, C., In Situ µGISAXS: I. Experimental Setup for Submicron Study of Protein Nucleation and Growth. Biophys. J. 2010, 99, 1256-1261. (3) Boyko, K. M.; Popov, V. O.; Kovalchuk, M. V., Promising approaches to crystallization of macromolecules suppressing the convective mass transport to the growing crystal. Russ. Chem. Rev. 2015, 84, 853-859. (4) McPherson, A.; Cudney, B., Optimization of crystallization conditions for biological macromolecules. Acta Crystallogr., Sect. F: Struct. Biol. Commun. 2014, 70, 1445-1467.


16

Page 17 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5) Castagnolo, D.; Vergara, A.; Paduano, L.; Sartorio, R.; Annunziata, O., Analysis of the influence of coupled diffusion on transport in protein crystal growth for different gravity levels. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 1633-1637. (6) Day, J.; McPherson, A., Macromolecular crystal growth experiments on International Microgravity Laboratory--1. Protein Sci. 1992, 1, 1254-1268. (7) Poodt, P. W. G.; Heijna, M. C. R.; Schouten, a.; Gros, P.; Enckevort, W. J. P. V., Simple Geometry for Diffusion Limited Protein Crystal Growth Harnessing Gravity to Suppress Convection. Cryst. Growth Des. 2009, 9, 885-888. (8) Littke, W.; John, C., Protein Single Crystal Growth Under Microgravity. Eur. Space Agency, [Spec. Publ.], SP 1984, 95, 185-188. (9) Fazio, V. J.; Peat, T. S.; Newman, J., A drunken search in crystallization space. Acta Crystallogr., Sect. F: Struct. Biol. Commun. 2014, 70, 1303-1311. (10) Chattopadhyay, S.; Erdemir, D.; Evans, J. M. B.; Ilavsky, J.; Amenitsch, H.; Segre, C. U.; Myerson, A. S., SAXS Study of the Nucleation of Glycine Crystals from a Supersaturated Solution. Cryst. Growth Des. 2005, 5, 523-527. (11) Myerson, A. S., Concluding remarks. Faraday Discuss. 2015, 179, 543-547. (12) Vorontsova, M.; Maes, D.; Vekilov, P. G., Recent advances in the understanding of twostep nucleation of protein crystals. Faraday Discuss. 2015, 4, 1166-1169. (13) Cui, H. L.; Yu, Y.; Chen, W. C.; Kang, Q., Study of Growth Mechanism of Lysozyme Crystal by Batch Crystallization Method. Chin. Chem. Lett. 2006, 17, 101-104. (14) Bonneté, F.; Ferté, N.; Astier, J. P.; Veesler, S., Protein crystallization: Contribution of small angle X-ray scattering (SAXS). J. Phys. IV 2004, 118, 3-13. (15) Vivarès, D.; Bonneté, F., X-ray scattering studies of Aspergillus flavus urate oxidase: towards a better understanding of PEG effects on the crystallization of large proteins. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 472-9. (16) Zhang, F.; Skoda, M. W. a.; Jacobs, R. M. J.; Martin, R. a.; Martin, C. M.; Schreiber, F., Protein interactions studied by SAXS: Effect of ionic strength and protein concentration for BSA in aqueous solutions. J. Phys. Chem. B 2007, 111, 251-259. (17) Ducruix, A.; Guilloteau, J. P.; Riès-Kautt, M.; Tardieu, A., Protein interactions as seen by solution X-ray scattering prior to crystallogenesis. J. Cryst. Growth 1996, 168, 28-39. (18) Bonneté, F.; Finet, S.; Tardieu, a., Second virial coefficient: variations with lysozyme crystallization conditions. J. Cryst. Growth 1999, 196, 403-414. (19) Pusey, M. L.; Snyder, R. S.; Naumann, R., Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals. J. Biol. Chem. 1986, 261, 6524-6529. (20) Wiechmann, M.; Enders, O.; Zeilinger, C.; Kolb, H. A., Analysis of protein crystal growth at molecular resolution by atomic force microscopy. Ultramicroscopy 2001, 86, 159-166. (21) Boué, F.; Lefaucheux, F.; Robert, M. C.; Rosenman, I., Small angle neutron scattering study of lysozyme solutions. J. Cryst. Growth 1993, 133, 246-254. (22) Heijna, M. C. R.; Van Enckevort, W. J. P.; Vlieg, E.; Enckevort, W. J. P. V., Growth Inhibition of Protein Crystals: A Study of Lysozyme Polymorphs. Cryst. Growth Des. 2008, 8, 270-274. (23) Feigin, L. A. A.; Svergun, D. I. I., Structure Analysis by Small-Angle X-Ray and Neutron Scattering. ed.; Plenum Press: New York, 1987; p 176-176. (24) Guilloteau, J.-P.; Rie`s-Kautt, M. M.; Ducruix, A. F., Variation of lysozyme solubility as a function of temperature in the presence of organic and inorganic salts. J. Cryst. Growth 1992, 122, 223-230.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 19

(25) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K., Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486-501. (26) Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.3r1. In ed.; 2010. (27) Kovalchuk, M. V.; Prosekov, P. A.; Marchenkova, M. A.; Blagov, A. E.; D’yakova, Y. A.; Tereshchenko, E. Y.; Pisarevskii, Y. V.; Kondratev, O. A., In situ study of the growth and degradation processes in tetragonal lysozyme crystals on a silicon substrate by high-resolution X-ray diffractometry. Crystallogr. Rep. 2014, 59, 679-684. (28) Korneev, V. N.; Shlektarev, V. A.; Zabelin, A. V.; Aul’chenko, V. M.; Tolochko, B. P.; Ariskin, N. I.; Lanina, L. F.; Vazina, A. A., X-ray stations based on cylindrical zoom lenses for nanostructural investigations using synchrotron radiation. J. Surf. Invest.: X-Ray, Synchrotron Neutron Tech. 2012, 6, 849-864. (29) Huang, T. C.; Toraya, H.; Blanton, T. N.; Wu, Y., X-ray powder diffraction analysis of silver behenate, a possible low-angle diffraction standard. J. Appl. Crystallogr. 1993, 26, 180184. (30) Hammersley, A. P., FIT2D: An Introduction and Overview. In 1997. (31) Konarev, P. V.; Volkov, V. V.; Sokolova, A. V.; Koch, M. H. J.; Svergun, D. I., : a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 2003, 36, 1277-1282. (32) Konarev, P. V.; Petoukhov, M. V.; Volkov, V. V.; Svergun, D. I., ATSAS 2.1, a program package for small-angle scattering data analysis. J. Appl. Crystallogr. 2006, 39, 277-286. (33) Svergun, D.; Barberato, C.; Koch, M. H., CRYSOL - A program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 1995, 28, 768-773. (34) Svergun, D. I.; Konarev, P. V.; Volkov, V. V.; Koch, M. H. J.; Sager, W. F. C.; Smeets, J.; Blokhuis, E. M., A small angle x-ray scattering study of the droplet–cylinder transition in oilrich sodium bis(2-ethylhexyl) sulfosuccinate microemulsions. J. Chem. Phys. 2000, 113, 16511665.


18

Page 19 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only Investigation of the initial crystallization stage in lysozyme solutions by small-angle X-ray scattering Mikhail V. Kovalchuk a,b,c, Alexander E. Blagovb,a, Yulia A. Dyakovab,a, Andrey Yu. Gruzinova, Margarita A. Marchenkova*,b,a, Georgy S. Petersa, Yury V. Pisarevskyb,a, Vladimir I. Timofeevb,a and Vladimir V. Volkovb,a a

National Research Centre “Kurchatov Institute”, 123182, Moscow, Russian Federation

b

Shubnikov Institute of Crystallography of Russian Academy of Sciences, 119333 Moscow,

Russian Federation c

St. Petersburg State University, 199034, St. Petersburg, Russian Federation


19

Investigation of the Initial Crystallization Stage in Lysozyme Solutions

Recommend Documents