JP6408321B2 - Method for producing protein crystal for X-ray crystal structure analysis - Google Patents

Description

  The present invention relates to a method for producing a protein crystal for X-ray crystal structure analysis. More specifically, the present invention relates to a method for producing a protein crystal for X-ray crystal structure analysis, which can promote protein crystallization without using a dedicated facility or the like.

  The function of a protein is closely related to its three-dimensional structure, and in order to understand the function of a protein, it is necessary to elucidate its three-dimensional structure. At present, X-ray crystal structure analysis is the only method for elucidating the three-dimensional structure of a protein having a molecular weight of tens of thousands or more. When using this method, it is necessary to crystallize the protein.

  However, there are proteins that are very difficult to produce crystals, and there is a problem of how to produce crystals of these proteins. Therefore, the solution of such a problem is desired.

  In the protein crystallization condition searching method of Patent Document 1, the crystallization condition can be efficiently searched by a step of mixing the protein crystallization agent and the solution in which the protein is dissolved.

  In the rapid crystallization method of the biopolymer species of Patent Document 2, the step of generating a concentrated solution of the biopolymer species using an electric field in the crystallization reaction device and the step of obtaining crystals in the crystallization reaction device, Large crystals can be obtained in a short time.

  In the method for producing crystal nuclei of Patent Document 3, crystal nuclei are generated by irradiating a solution in which a solute to be crystallized is irradiated with a pulsed laser.

  In Patent Document 4, regarding the structural analysis of nanomaterials such as metal microparticles and polymer microparticles, the protein is crystallized in a state where the nanomaterial to be analyzed and the protein coexist in a solvent, and the protein crystals are refined. It has been proposed that a nanomaterial aggregate is obtained by integrating nanomaterials in pores and analyzed by X-ray crystal structure analysis. Here, by using a known protein as this protein, it is said that the diffraction scattering image of the nanomaterial can be obtained by subtracting the diffraction scattering image of the known protein from the diffraction scattering image of the nanomaterial aggregate. Yes.

US Patent Application Publication No. 2013-0344523 US Patent Application Publication No. 2009-0101491 International Publication No. 2004-018744 Specification JP 2011-31333 A

  In the protein crystallization condition searching method of Patent Document 1, there is a possibility that proteins are aggregated irregularly by a protein crystallization agent.

  In the rapid crystallization method of the biopolymer species of Patent Document 2, an electric field is used, so that dedicated equipment, materials, and conditions are required.

  In the method for producing crystal nuclei in Patent Document 3, a laser is used, so that dedicated equipment, materials, and conditions are required.

  The method for producing a nanomaterial assembly of Patent Document 4 is for structural analysis of nanomaterials such as metal fine particles and polymer fine particles.

  Accordingly, an object of the present invention is to provide a method for producing a protein crystal for X-ray crystal structure analysis, which can promote protein crystallization without using a dedicated facility or the like.

The present invention is as follows.
<1> including crystallizing the protein from the solvent, the fine particles or clusters dispersed in the solvent, and the solution containing the protein to be evaluated by X-ray crystal structure analysis using the fine particles or clusters as crystal nuclei. A method for producing a protein crystal for X-ray crystal structure analysis.
<2> The production method according to <1>, wherein the fine particles or clusters are metal fine particles or metal clusters.
<3> The production method according to <1> or <2>, wherein the solution contains a stabilizer that stabilizes dispersion of the fine particles or clusters in the solvent.
<4> The production method according to <3>, wherein the stabilizer is selected from the group consisting of a carboxylic acid, a surfactant, and a polymer compound.
<5> A protein crystal for X-ray crystal structure analysis, including fine particles serving as crystal nuclei and a protein to be evaluated by X-ray crystal structure analysis.
<6> The protein crystal according to <1>, wherein the fine particles or clusters are metal fine particles or metal clusters.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the protein crystal | crystallization for X-ray crystal structure analysis which can accelerate | stimulate crystallization of protein can be provided, without using an exclusive installation etc.

  Hereinafter, embodiments of the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist of the present invention.

<< Method for producing crystallized protein for X-ray crystal structure analysis >>
The present invention is a method for producing a protein crystal for X-ray crystal structure analysis.

  In the method of the present invention for producing a protein crystal for X-ray crystal structure analysis, fine particles or clusters are extracted from a solvent, fine particles or clusters dispersed in the solvent, and a solution containing the protein to be evaluated by X-ray crystal structure analysis. Crystallizing proteins as crystal nuclei.

  In general, protein crystallization is composed of two stages of crystal nucleation and crystal growth. Protein crystal nuclei are formed in regions with a high protein concentration, but crystal nuclei are unstable, and do not become stable unless they have a certain size. Therefore, in conventional protein crystallization methods, crystal nuclei are difficult to form or are not formed, and thus protein crystals may not be produced.

  On the other hand, in the method of the present invention for producing a protein crystal for X-ray crystal structure analysis, fine particles or clusters are dispersed in a solvent, so that the fine particles or clusters and the protein interact and are formed from them. The frequency of appearance of crystal nuclei increases, and protein crystallization can be promoted.

  The interaction between the fine particle or cluster and the protein is an interaction due to a complex factor including an electrostatic interaction and a hydrophobic interaction. This includes the interaction of proteins with stabilizers present around the microparticles or clusters. This interaction generates a locally high region of protein concentration around the microparticles or clusters, which promotes nucleation. In addition to nucleation within this complex, crystal nucleation by fusion of multiple complexes is also conceivable. In addition, the surface potential of the protein is perturbed by the surface potential of the fine particles or clusters in this complex, and irregular aggregation is suppressed, and at the same time, nucleation by fusion of the complex or multiple complexes is promoted.

  Furthermore, in the method of the present invention for producing a protein crystal for X-ray crystal structure analysis, a dedicated process or the like is not used, so that a simple process, low cost, and low environmental load can be realized.

(Fine particles)
In the solution used in the method of the present invention, fine particles or clusters are dispersed in a solvent. The fine particles or clusters can be dispersed in the solvent by any method.

  In order to disperse the fine particles or clusters in the solvent, for example, by adjusting the pH or the like to control the charge state of the fine particles or the cluster surface, the fine particles or clusters are repelled to each other and their aggregation is prevented. Alternatively, the clusters can be dispersed in a solvent.

  The charge state on the surface of the fine particle or cluster cannot be directly measured, but can be indirectly known by measuring the zeta potential (electrokinetic potential). In general, when the absolute value of the zeta potential is large, the charge density on the surface of the fine particles is high, whereby the fine particles repel each other. On the other hand, when the absolute value of the zeta potential is small, the charge density on the surface of the fine particles or clusters is low, whereby the fine particles or clusters tend to aggregate.

  For example, the zeta potential of Pt microparticles greatly depends on the pH, and the zeta potential slightly increases with decreasing pH when the pH is 8 or less, whereas when the pH is higher than 8, the zeta potential increases. It decreases rapidly with.

This is because the platinum atoms on the surface of the fine particles are partially oxidized, and as the pH decreases, the oxidized platinum atoms become Pt-OH, and at the same time, protons are added to some of the Pt atoms on the surface of the Pt fine particles. This is because the zeta potential increases as the density of positive charges increases due to Pt-H ⁺ . On the other hand, since the oxidized platinum atom becomes Pt—O ⁻ with the increase in pH, and the portion of the surface of the Pt fine particles is deprotonated to reduce the density of positive charges, the zeta potential is lowered. Conceivable.

  Moreover, in order to disperse the fine particles or clusters in the solvent, the following stabilizer can be contained in the solvent.

The average particle size of the fine particles or clusters can be 1000 nm or less, 500 nm or less, 100 nm or less, 50 nm or less, 20 nm or less, 10 nm or less, or 1 nm or less in order to improve the dispersibility of the fine particles in the solvent. In the present invention, particles having a size of 1 nm or more are referred to as “fine particles”, and particles having a size of less than 1 nm are referred to as “clusters”. The average particle diameter of the fine particles can be 1 nm or more, or 5 nm or more in order to improve the handleability of the fine particles. The average particle size of the fine particles can be measured using, for example, a dynamic light scattering measurement device (ELSZ-1000Z type) manufactured by Otsuka Electronics.

  The concentration of the fine particles or clusters may be appropriately determined according to the pH of the solution, the solubility and concentration of the protein, the type of solvent, the crystallization temperature, and the like, and is not particularly limited. For example, the concentration of the fine particles should be in the range of 1 nM to 200 nM, preferably 10 nM to 150 nM, more preferably 15 nM to 100 nM, and particularly preferably 20 nM to 80 nM in order to promote protein crystallization on the basis of the number. Can do.

  As the fine particles or clusters, any fine particles or clusters can be used, and examples thereof include metal fine particles or metal clusters, non-metallic fine particles or non-metallic clusters, or polymer fine particles or polymer clusters.

  The metal fine particles or metal clusters are not particularly limited as long as they are fine particles or clusters of typical metals and transition metals. Examples of typical metal fine particles or clusters include Li, Be, Na, Mg, Al, K, Ca, Zn, Ga, Ge, Rb, Sr, Cd, In, Sn, Sb, Cs, Ba, Hg, TI, Pb, Bi, Po, Fr, Ra, etc. can be mentioned. Examples of the transition metal fine particles or clusters include Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, lanthanoid series, and Hf. , Ta, W, Re, Os, Ir, Pt, Au, actinoid, Rf, Db, Sg, Bh, Hs, Mt, and the like. Among the transition metal fine particles or clusters, Au, Ag, Ru, Rh, Pd, Os, Ir, Pt, Cu, Fe, Ni, Co, Cr, Mn, Mo, W, Ta, and Nb are more preferable. , Au, Ag, and platinum group (Ru, Rh, Pd, Os, Ir, Pt) and the like are more preferable from the viewpoint of being hardly oxidized, and Au and Pt are particularly preferable. Further, fine particles or clusters of composite metal such as GaAs, GaTe, CdSe may be used.

  As a method for producing metal fine particles or clusters, a method of ablating a metal plate surface by laser irradiation or microwave irradiation in a liquid such as water, a metal plate surface in a liquid such as water to which a surfactant is added, or A method of ablation by microwave irradiation, a method of chemical reduction, a method of discharging in a solution, and the like are mentioned, and there is no particular limitation.

  Non-metallic fine particles or non-metallic clusters include fine particles or clusters of organic compounds such as organic dyes and organic pigments, or inorganic compounds such as inorganic pigments.

  Examples of the polymer fine particles or polymer clusters include fine particles or clusters such as polystyrene, polyethylene, polypropylene, or latex.

(Stabilizer)
As described above, in order to disperse the fine particles or clusters in the solvent, a stabilizer can be contained in the solvent.

  In the absence of a stabilizer in the solvent, the fine particles or clusters are energetically unstable due to low affinity with the solvent, and the like. Or it may precipitate.

  On the other hand, when a stabilizer is present in the solvent, the stabilizer is coordinated to the surface of the fine particles or clusters, thereby preventing the aggregation or precipitation of the fine particles or clusters, and the fine particles or clusters in the solvent. Dispersion can be stabilized.

  When the stabilizer is an amphiphilic nonionic substance, it hardly affects the surface charge of the fine particles or clusters, if any.

  The concentration of the stabilizer may be appropriately determined according to the pH of the solution, the concentration of the protein, the type of solvent, the crystallization temperature, and the like, and is not particularly limited, but the fine particles or clusters are dispersed in the solvent. 10 mg / mL or more, 50 mg / mL or more, 100 mg / mL or more, 150 mg / mL or more, 200 mg / mL or more, 300 mg / mL or more, 500 mg / mL or more, or 1000 mg / mL or more, From the viewpoint of promoting the interaction between microparticles or clusters and proteins, at 10000 mg / mL or less, 5000 mg / mL or less, 3000 mg / mL or less, 1000 mg / mL or less, 500 mg / mL or less, 300 mg / mL or less, or 100 mg / mL or less. Good.

  As the stabilizer, any stabilizer can be used as long as the dispersion of the fine particles or clusters in the solvent can be stabilized. For example, a carboxylic acid stabilizer, a surfactant stabilizer, or a high stabilizer can be used. Examples thereof include molecular compound stabilizers.

  Examples of the carboxylic acid stabilizer include citric acid or oxalic acid stabilizer.

  As the surfactant, an anionic, cationic, nonionic or amphoteric surfactant can be used. For example, sodium dodecyl sulfate (SDS) or oleic acid stabilizer Can be mentioned.

  Examples of the polymer compound stabilizer include polyvinyl pyrrolidone (PVP) or polyvinyl pyridine stabilizer.

(Protein to be evaluated by X-ray crystal structure analysis)
Proteins to be evaluated by X-ray crystal structure analysis are contained in a solution containing a solvent and fine particles or clusters dispersed in the solvent. Furthermore, an optional protein solubility modifier that adjusts the solubility of the protein in the solution may be added to the solution to facilitate crystallization of the protein.

  Proteins to be evaluated by X-ray crystal structure analysis can be arbitrarily selected.

  The protein concentration to be evaluated by X-ray crystal structure analysis may be appropriately determined according to the concentration of fine particles or clusters, the pH of the solution, the solubility of the protein, the type of solvent, the crystallization temperature, and the like, and is particularly limited. However, if the molecular weight of the protein is high, considering that the number of protein molecules in the solution decreases, 1 mg / mL or more, 5 mg / mL or more, 10 mg / mL or more, 50 mg / mL or more, 100 mg / mL From the viewpoint of facilitating the formation of crystal nuclei by microparticles or clusters and protein, in order to ensure that the protein is sufficiently present around the microparticles, 10,000 mg / mL or more may be 150 mg / mL or 200 mg / mL or more. mL or less, 5000 mg / mL or less, 3000 mg / mL or less, 1000 mg / mL or less, 500 mg mL or less, 300 mg / mL or less, or 100 mg / mL or less.

  The protein solubility regulator to be evaluated by X-ray crystal structure analysis may be appropriately selected according to the concentration of fine particles or clusters, the pH of the solution, the concentration and solubility of the protein, and the crystallization temperature. Examples thereof include inorganic salts such as sodium chloride (NaCl) and ammonium sulfate, alcohols such as polyethylene glycol (PEG) and ethanol, and nonionic surfactants such as NP-40 (polyoxyethylene (9) octylphenylether).

  The concentration of the protein solubility regulator to be evaluated by X-ray crystal structure analysis may be determined as appropriate according to the concentration of fine particles or clusters, the pH of the solution, the concentration and solubility of the protein, the crystallization temperature, etc. There is no.

  The compatibility between the protein solubility regulator and the stabilizer to be evaluated in the X-ray crystal structure analysis is appropriately determined according to the concentration of fine particles or clusters, the pH of the solution, the concentration and solubility of the protein, and the crystallization temperature. Although there is no particular limitation, for example, from the viewpoint of increasing the dispersibility of Pt fine particles, the compatibility between sodium chloride and PVP is good.

(solvent)
The solvent is contained in a solution containing fine particles or clusters and a protein to be evaluated by X-ray crystal structure analysis.

  The type of the solvent is not particularly limited as long as it dissolves proteins and dissolves fine particles or clusters or disperses them as uniformly as possible. Water or a general organic solvent can be used.

  Water is not particularly limited, and in order to promote crystallization, it is better that there are few impurities. Usually, pure water such as ion-exchanged water or ultrapure water is used.

  Examples of the organic solvent include alcohol solvents such as methanol, ethanol, and isopropyl alcohol; aromatic solvents such as benzene and toluene; halogen solvents such as methylene chloride, chloroform, and carbon tetrachloride; Chain saturated hydrocarbon solvents such as n-hexane and n-heptane; cyclic saturated hydrocarbon solvents such as cyclohexane; and nitrile solvents such as acetonitrile can be used. Among these, water and alcohol-based solvents are preferable because of wide application range, and water is more preferable.

(Crystalline protein for X-ray crystal structure analysis)
Crystallized proteins for X-ray crystal structure analysis crystallize proteins using fine particles or clusters as crystal nuclei.

  As a method for crystallizing protein, any crystallization method may be used, and examples thereof include a vapor diffusion method, a batch method, a liquid-liquid diffusion method, a dialysis method, and a seeding method.

  Examples of the vapor diffusion method include a hanging drop method and a sitting drop method.

  The hanging drop method is a method in which a droplet of a solution containing protein and fine particles is attached to a cover glass and suspended, and is vapor-balanced in a closed system with respect to a reservoir solution of about 100 times its capacity. .

  The sitting / drop method is a method in which a droplet of a solution containing protein and fine particles is allowed to stand in a recess and is vapor-balanced in a closed system with respect to a reservoir solution having a volume about 100 times that of the solution.

  As the reservoir solution, a solution having the same composition as the solution excluding proteins and fine particles or clusters or a solution having a final target composition may be used.

  The crystallization temperature may be determined according to the ease of crystallization of the protein to be used, the concentration of the protein and fine particles in the crystallization solution, the concentration of the protein solubility adjusting agent, and the like. There is no particular limitation as long as the temperature is such that no decomposition occurs. For example, it is the range of 0 degreeC-90 degreeC.

  The time for crystallization may be determined according to the ease of crystallization of the protein to be used, the concentration of the protein and nanomaterial in the crystallization solution, the concentration of the protein solubility adjusting agent, etc., and is not particularly limited. . For example, it is about 60 minutes to 10,000 hours.

<< Crystalline protein for X-ray crystal structure analysis >>
The protein crystal for X-ray crystal structure analysis of the present invention includes fine particles or clusters serving as crystal nuclei and a protein to be evaluated by X-ray crystal structure analysis.

  Protein crystals for X-ray crystal structure analysis can be used in drug discovery research such as SBDD (Structure-Based Drug Design).

  The present invention will be described in more detail with reference to the following examples, but it goes without saying that the scope of the present invention is not limited by these examples.

Example 1
<Preparation of protein solution>
A protein solution was prepared by dissolving lysozyme as a protein and sodium chloride (NaCl) as a protein solubility adjuster in pure water as a solvent.

<Preparation of fine particle solution>
55 mg of PVP as a stabilizer was dissolved in 5 mL of pure water as a solvent. This solution was placed in a container, and a Pt metal plate was placed at the bottom of the container. This metal plate was ablated by irradiating a pulse laser having a wavelength of 1064 nm and a pulse width of 10 nS, and Pt fine particles were monodispersed in the solution to prepare a Pt fine particle solution.

<Measurement of zeta potential>
The zeta potential of the Pt fine particles was measured. According to this, when the pH is 8 or more, the absolute value of the zeta potential of the Pt microparticles greatly increases as the pH increases, but when the pH is 8 or less, the absolute value of the zeta potential of the Pt microparticles is small. At 3.0, the zeta potential was about 1 mV.

<Protein crystallization>
The protein solution and the fine particle solution were mixed to obtain a mixed solution, and protein was crystallized by using a sitting drop method on the mixed solution. In this mixed solution, the concentration of lysozyme as protein was 14.3 mg / ml, the concentration of NaCl as a protein solubility regulator was 4% by mass, the pH of the solution was 4, and the concentration of fine particles was 13.2 nM. Met.

<Evaluation>
When crystallization was performed three times, an average of 9.5 protein crystals were obtained.

<< Examples 2 to 6 >>
Protein crystallization was performed in the same manner as in Example 1 except that the concentration of fine particles in the mixed solution was changed as shown in Table 1 below.

<< Comparative Example 1 >>
The protein was crystallized in the same manner as in Example 1 except that the fine particle solution was not used, and therefore the fine particles and the stabilizer were not used.

<< Comparative Example 2 >>
Pt fine particles in which PVP adhered only to the particle surface were prepared. This was repeated three times using a centrifuge to precipitate Pt fine particles and wash the Pt fine particles by redispersion in pure water. Protein crystallization was performed in the same manner as in Example 1 except that no further stabilizer was used.

<result>
Table 1 below shows an outline of the experimental conditions of Examples 1 to 6 and Comparative Examples 1 and 2 and the evaluation results.

<< Examples 8 to 13 >>
Protein crystallization was performed in the same manner as in Example 1 except that the concentration of lysozyme was 13.0 mg / ml and the concentration of fine particles in the mixed solution was changed as shown in Table 2 below. .

<< Comparative Example 3 >>
The protein was crystallized in the same manner as in Example 8 except that the concentration of lysozyme was 13.0 mg / ml, the fine particle solution was not used, and therefore no fine particles were used.

<result>
Table 2 below shows an outline of the experimental conditions and the evaluation results of Examples 8 to 13 and Comparative Example 3.

Tables 1 and 2 show that the average value of the number of lysozyme crystals increases in proportion to the concentration of Pt fine particles. In particular, this tendency is remarkable from Table 1 showing that the concentration of lysozyme is high. Further, when Examples 1 to 6 and Comparative Example 1 in Table 1 are compared, it can be seen that Examples 1 and 3 to 6 exceed the average value of the number of crystals compared to Comparative Example 1. Furthermore, even if Examples 8-13 of Table 2 and Comparative Example 3 are compared, it turns out that it is the same result.

  This result is considered to be because the Pt fine particles and lysozyme interact with each other due to the dispersion of the Pt fine particles, the frequency of appearance of crystal nuclei formed from them increases, and the crystallization of lysozyme is promoted.

  Furthermore, in Comparative Example 2 in Table 1, the average value of the number of lysozyme crystals is zero. This is because, when PVP is coordinated only on the surface of the Pt fine particles, the Pt fine particles in the solution become unstable in energy (for example, surface energy), so that an aggregate of Pt fine particles or Pt fine particles And lysozyme aggregates were formed to inhibit lysozyme crystallization.

  Therefore, it can be seen that the dispersibility of the fine particles, particularly Pt fine particles, is an important factor for promoting crystallization of proteins, particularly lysozyme crystals.

  Furthermore, in order to clarify the mechanism of crystal nucleation, dynamic light scattering of a crystal solution containing Pt fine particles and lysozyme molecules was measured. As a result, it was revealed that Pt fine particles and lysozyme molecules formed a complex. In this complex, it is considered that a region having a high lysozyme molecule concentration is formed around the Pt fine particles, and crystal nucleation is promoted.

  We also measured the zeta potential and clarified the relationship between the surface potential and the promotion of crystal nucleation. According to this, it was found that the zeta potential was 1 mV only for Pt fine particles and 6 mV only for lysozyme molecules, but the complex of Pt fine particles and lysozyme molecules was 2.5 mV. This is presumably because the formation of a complex reduces the surface potential of lysozyme having a surface potential higher than that of the Pt fine particles, thereby suppressing the repulsion between lysozyme molecules, thereby promoting the formation of crystal nuclei. In this case, formation of crystal nuclei by fusion of a complex or a plurality of complexes can be considered.

  Although preferred embodiments of the present invention have been described in detail, proteins, protein solubility modifiers, microparticles or clusters, stabilizers, solvents, and crystals used in the present invention without departing from the scope of the claims Those skilled in the art will appreciate that various modifications can be made to the formulation.

Claims (8)

X-ray comprising crystallizing the protein from the solvent, fine particles or clusters dispersed in the solvent, and a solution containing the protein to be evaluated by X-ray crystal structure analysis using the fine particles or clusters as crystal nuclei. A method for producing a protein crystal for crystal structure analysis. The manufacturing method according to claim 1, wherein the fine particles or clusters are metal fine particles or metal clusters. The manufacturing method of Claim 1 or Claim 2 in which the said solution contains the stabilizer which stabilizes dispersion | distribution of the said microparticles | fine-particles or cluster in the said solvent.   The production method according to claim 3, wherein the stabilizer is selected from the group consisting of a carboxylic acid, a surfactant, and a polymer compound. Producing a protein crystal for X-ray crystal structure analysis by the method according to any one of claims 1 to 4, and
Performing X-ray crystal structure analysis on the protein crystal for X-ray crystal structure analysis, and analyzing the three-dimensional structure of the protein constituting the protein crystal for X-ray crystal structure analysis,
A method for analyzing an X-ray crystal structure, comprising: A protein crystal for X-ray crystal structure analysis comprising fine particles or clusters serving as crystal nuclei and a protein to be evaluated by X-ray crystal structure analysis. The protein crystal according to claim 6 , wherein the fine particles or clusters are metal fine particles or metal clusters. The protein crystal for X-ray crystal structure analysis according to claim 6 or 7 is subjected to X-ray crystal structure analysis to analyze a three-dimensional structure of a protein constituting the protein crystal for X-ray crystal structure analysis. A method for analyzing an X-ray crystal structure, comprising: