Nanodiamonds as Nucleating Agents for Protein Crystallization

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Nanodiamonds as Nucleating Agents for Protein Crystallization Yen-Wei Chen, Chien-Hsun Lee, Yung-Lin Wang, Tsung-Lin Li, and Huan-Cheng Chang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00578 • Publication Date (Web): 11 Jun 2017 Downloaded from http://pubs.acs.org on June 17, 2017

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Nanodiamonds as Nucleating Agents for Protein Crystallization

Yen-Wei Chen,1,† Chien-Hsun Lee,1,† Yung-Lin Wang,2 Tsung-Lin Li,2 and Huan-Cheng Chang*,1,2,3

1

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan

2

Genomics Research Center, Academia Sinica, Taipei 115, Taiwan

3

Department of Chemical Engineering, National Taiwan University of Science and

Technology, Taipei 106, Taiwan †

These two authors contributed equally to this work.

*E-mail: [email protected]

ABSTRACT Nanodiamond (ND) is a carbon-based nanomaterial with potential for a wide range of biological applications.

One of such applications is to facilitate the nucleation of protein

crystals in aqueous solution.

Here, we show that NDs (nominal diameters of 30 and 100 nm)

after surface oxidation in air and subsequent treatment in strong acids are useful as heterogeneous nucleating agents for protein crystallization.

Tested with lysozyme,

ribonuclease A, proteinase K, and catalase, the nanomaterials in either aggregate or film form are found to be able to increase the crystallization efficiency of all proteins.

Particularly, for

30 nm NDs, the films with an area of ~2 mm2 can effectively induce the crystallization of lysozyme at a concentration as low as 5 mg/mL.

The efficiency can be further improved by

1 ACS Paragon Plus Environment

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adding pre-formed protein clusters (~300 nm in diameter) as inherent nucleation precursors, as demonstrated for ribonuclease A.

This combined approach is easy to implement, highly

compatible with existing technologies, and can be applied to other protein samples as well.


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INTRODUCTION Crystallization is a critical step in determining the three-dimensional structure of a protein by X-ray crystallography.1

However, due to the complexities of crystal formation mechanism

and the difficulties of obtaining high-quality protein crystals, it often represents a bottleneck in structure determination.2 crystallization process.

Heterogeneous nucleation plays a central role in the protein

It occurs when heterogeneous substrates such as horse hair and

human hair3-5 are added to the crystallization buffers to lower the nucleation energy barrier. A number of nanomaterials such as porous silicon, gold nanoparticles, silica beads, carbon nanotubes, and graphene have been proposed and tested as nucleants to enhance the heterogeneous nucleation and subsequent crystal growth.6-11

However, to date, there have

been no universal nucleants for protein crystallization, although nanoporous materials appear to be a promising candidate.12,13

Govada et al.14 have recently conducted an extensive study

on carbon nanomaterial diversity for protein crystal nucleation.

Among various

surface-modified carbon nanotubes, graphene oxides, and carbon black tested, the most effective system is the carbon black conjugated with poly(ethylene glycol) methyl ether of 5 kDa in molecular weight. This work explores the feasibility of using surface-oxidized diamond nanoparticles to assist protein crystallization in aqueous solution. (NDs) are many-fold.

The reasons to choose nanodiamonds

First, NDs can be synthesized by detonation, high-pressure

high-temperature, and chemical vapor deposition methods in large scale and they are available in various sizes from 5 nm to tens micrometers.15

Second, NDs can be easily

purified by strong acid washes to remove all possible impurities and contaminants,16 a step of critical importance in producing high-quality protein crystals.

Third, the surface properties

of NDs are tunable from hydrophilic to hydrophobic, or vice versa, after proper chemical modifications.17

Fourth, NDs after strong oxidative acid washes have an exceptionally high


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affinity for proteins of various types18 and both water-soluble and membrane proteins can be extracted by NDs in the presence of detergents like sodium dodecyl sulfate.19-21

Finally,

porous ND films of different dimensions (area and thickness) can be readily fabricated by depositing NDs on commonly used protein crystallization plates and the protocol is highly compatible with vapor diffusion and other crystallization methods.22 How the substrate topography and surface chemistry of the nanomaterials affect the protein crystallization processes can be closely examined for further improvement.23-26 The applications of NDs as nucleants for protein crystallization in this work consist of two parts: (1) ND aggregates and (2) ND films.

The first application follows conventional

procedures by adding surface-oxidized NDs directly into protein crystallization buffers in the individual trials of crystallization screens.13

The NDs form porous submicron aggregates

rapidly in the high ionic strength medium due to the weakening of repulsive forces between the charged nanoparticles.

The second approach is motivated by the Monte Carlo

simulations of Sear and coworkers,27,28 who predicted that the nucleation rates of protein molecules at pores are several orders of magnitude higher that on flat surfaces.

An

advantage of this approach is that it reduces the effect of solution turbulence on the protein crystallization,22 if heterogeneous nucleation occurs at the pores of the films firmly attached to a solid substrate.

Another benefit of this approach is that the size of the ND film can be

made as small as needed to limit the number of nuclei formed during crystallization,2 which in turn can lead to the growth of fewer crystals of larger size.

The method is expected to

facilitate heterogeneous nucleation at low protein concentrations and enhance crystal growth rates. Four proteins were tested against the surface-oxidized NDs of two different sizes (nominal diameters of 30 nm and 100 nm) and two different configurations (aggregates and films): lysozyme, ribonuclease A (RNase A), proteinase K, and catalase. We started the


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experiment by using a commercial crystallization screening kit to investigate whether the presence of porous ND aggregates can assist the crystal formation of these four proteins in 98 trails.

Next, experiments with lysozyme as the model protein were conducted to compare

the performance of NDs as nucleants in either aggregate or film form under same crystallization conditions.

Finally, we explored the feasibility of using pre-formed protein

clusters in combination with ND films as the nucleating agents to assist the crystallization of RNase A at low concentrations.

MATERIALS AND METHODS Preparation of surface-oxidized NDs. Diamond powders with nominal sizes of 100 nm (Micron + MDA 0 – 0.1 µm, Element Six) and 30 nm (MSY 0 – 0.05 µm, Microdiamant) were treated in a strong oxidative acid mixture, H2SO4 and HNO3 (3:1, v/v ), in a microwave reactor (Discover BenchMate, CEM) to remove metallic impurities and graphitic structure on surface, as previously described.29

Number-averaged hydrodynamic diameters and size

distributions of the particles after extensive wash with deionized distilled water (ddH2O) were determined by dynamic light scattering (DLS) using a combined particle size and zeta potential analyzer (Delsa Nano C, Beckman-Coulter).

A scanning electron microscope

(JSM-7800F Prime, JEOL) characterized the size, shape, and morphology of the nanoparticles deposited on glass substrates.

Preparation of protein clusters.

Clustering of protein molecules was induced by

adding 3 M (NH4)2SO4 into the solution of interest (5 mg/mL).

The clustering process was

monitored in situ using a combined particle size and zeta potential analyzer (ZetaSizer Nano ZS, Malvern Instruments) by DLS.

Protein crystallization with ND aggregates. Four model proteins were obtained as highly purified lyophilized powders from Sigma Aldrich: lysozyme (L6876) from hen egg


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white, RNase A (R6513) from bovine pancreas, proteinase K (P6556) from Tritirachium

album, and catalase (C1345) from bovine liver.

To study the effect of ND aggregates on

protein crystallization, 98 precipitant solutions in combination with 2 differently sized NDs (100 or 30 nm in size) were used for the 4 proteins.

Commercial crystal screen kits

(Hampton Research Crystal Screen I and II, HR2-110 and HR2-112) served as the sources of the precipitant solutions and Greiner CrystalQuick 96-well sitting drop plates (Hampton Research HR8-148) acted as the crystal cultivation plates.

The experiment began with

loading of 100 µL precipitant solutions into the individual wells of the cultivation plates as reservoirs.

Protein drops (3 µL), each containing 50 µg/mL ND (100 nm or 30 nm) in the

precipitant solution, were then added on the plates.

Concentrations of the proteins used in

the trials were 25 mg/mL for lysozyme, 20 mg/mL for RNase A, 10 mg/mL for proteinase K, and 20 mg/mL for catalase.

Images of the protein crystals formed were acquired by using a

vertical optical microscope (DM 2700M, Leica) equipped with a color CCD camera (DFC 7000T, Leica) at various time points.

Preparation of ND films. Aliquots (5 µL each) of the Piranha solution (concentrated H2SO4:30% H2O2 = 3:1, v/v) were dropped in the individual wells of the sitting drop plates (Hampton HR3-160) for 2 h.

After being cleaned with ddH2O and dried in air, each well

was added with 1 µL of 3 mg/mL poly-L-lysine (Sigma P9404) solution on the Piranha-treated spot for surface coating with the cationic polymers for 3 h.

Finally, 5 µL of

the 100-nm (or 30-nm) ND solution prepared in ddH2O (concentration of 50 – 1000 µg/mL) was pipetted to the well and let dry overnight to form a porous film.

Protein crystallization with ND films.

Two precipitant solutions were used to

examine the effect of ND films on the protein crystallization: (1) 1.2 M NaCl, 50 mM CH3COONa, pH 4.5 for lysozyme and (2) 3 M NaCl, 1.2 M (NH4)2SO4, 50 mM CH3COONa, pH 5.5 for RNase A.


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X-ray crystallography. X-ray diffraction was carried out at 110 K with the addition of 20% glycerol as a cryoprotectant for samples using the beamline BL15A1 at the National Synchrotron Radiation Research Center, Taiwan.30

Diffraction data were collected with a

CCD detector (MX300-HE, Rayonix) for the lysozyme crystals grown in the presence of 30-nm and 100-nm ND films at a wavelength of 1 Å and processed to 1.41 Å and 1.79 Å resolution, respectively, using the HKL-2000 software.31

Molecular replacement (MR)

searches were performed with Phaser and the PDB entry 1LSG as a template.32

The MR

solution was subjected to restrained refinement in Phenix.33

RESULTS ND aggregates as nucleating agents.

The diamond powders used in this work were

synthesized by high-pressure-high-temperature methods.

They are monocrystalline and

their surface is modified with various oxygen-containing groups, including –COOH, –COH, and –C=O, after strong oxidative acid treatment.16

Our previous studies have characterized

in detail the surface properties of these surface-oxidized NDs and found a zeta potential in the range of −40 mV at pH ≥ 5, along with an isoelectric point of pI ~ 3.34

Uniquely, the

particles have an exceptionally high affinity for biomolecules such as polypeptides and proteins, made possible by the interplay of electrostatic attraction, hydrogen bonding, van der Waals interaction, and hydrophobic interactions between adsorbate and surface.18 Specifically, the surface of NDs can be rapidly saturated with protein molecules (such as lysozyme) at the concentration as low as 10 µg/mL in water.18,34

This high affinity renders

them useful as a solid extraction support to isolate and concentrate proteins of various types in physiological medium.19

For NDs of 100 nm (or 30 nm) in diameter, more than 1000 (or

100) globular protein molecules (such as myoglobin, cytochrome c, and lysozyme) can be closely packed on the surface at full coverage.34,35

This high density (~1 × 1014


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molecules/cm2) implies that protein-loaded NDs are potentially useful as a seed to initiate protein crystallization. To test the hypothesis, we used 30-nm and 100-nm acid-treated NDs as the nucleants for protein crystallization screens.

This was made by adding the nanoparticles directly into

the crystallization buffers containing various precipitants prepared from Hampton screen kit I and II. Both NDs formed aggregates with a hydrodynamic size of more than 200 nm at the particle concentration of 50 µg/mL in the buffers (Figure 1a and 1b).

While some were

freely suspended in the crystallization drops, most of them were precipitated within 1 – 2 days.

Using the vapor diffusion method in a hanging drop format, we performed the protein

crystallization experiments using lysozyme, RNase A, proteinase K, and catalase in the 98 trials with or without NDs.

It was found that, irrespective of the ND size, the mean numbers

of the trials with the protein crystals formed in the ND-treated groups (50 µg/mL) are all significantly higher than those in the control groups (Figure 2).

It suggests that both NDs in

their aggregate forms can facilitate the crystal growth of all four model proteins.

The size

effect of the nucleants, however, is irregular, varying with the type of the proteins used in the experiments.

ND films as nucleating agents.

Figure 3a shows a schematic diagram of the ND

film preparation described in the experimental section.

Briefly, a drop of the Piranha

solution was placed on a sitting drop plate made of polystyrene to create a small hydrophilic area.

Poly-L-lysine was then added to the acid-treated area to serve as the interface for

subsequent binding with surface-oxidized NDs.

The tight binding between the negatively

charged carboxylated nanoparticles and the positively charged amine-terminated plate by electrostatic forces and hydrogen bonding produced a sturdy ND film.16 Scanning electron microscopy (SEM) revealed that the particles making of the films are irregular in shape and heterogeneous in size (Figure 3b and 3c).

The film is highly porous, with the pore sizes


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varying over a wide range.

These pores may act as the nucleation sites for protein

crystallization. Based on the result presented in the earlier section that the ND aggregates can promote protein crystal growth, we expect that the porous ND films can similarly lower the energy barrier of the nucleation in protein crystallization.

In this approach, both the thickness and

area of the films are adjustable by changing the concentration of the ND suspensions as well as the volume of the Piranha solution that determines the film size.

For instance, the

diameter (area) of the film can be effectively reduced to ~1.5 mm (~2 mm2) by decreasing the volume of the Piranha solution to 1 µL (Figure 3d and 3e).

The reduction of the film area

helps localize the sites of crystal growth, reduce the numbers of crystals formed, and provide a more stable platform for protein crystallization.

Figure 4 compares the crystallization-promoting abilities of 100-nm ND films (a – d) and 30-nm ND films (e – h) at the particle concentrations of 0, 50, 250, and 500 µg/mL. Obviously, many more lysozyme crystals were formed in the presence of the thicker films. The effect is particularly prominent in the cases of the 30-nm ND films.

The result can be

rationalized by considering that smaller NDs have larger surface-to-volume ratios and so the films made of these particles are more porous than the 100 nm ones. Moreover, smaller particles provide a larger protein adsorption capacity per unit weight and thus have higher protein crystallization efficiency.

In the followings, to avoid making the films too thick to

hinder our microscopic examinations, we prepared the substrates with 500 µg/mL NDs.

The

average thickness of the films prepared in this manner is ~0.7 µm, assuming a porosity of 50%.

The pore diameters, as revealed by SEM (Figure 3b and 3c), are comparable to the

sizes of the constituent nanoparticles. We next investigated whether the ND films have the ability to induce the formation of lysozyme crystals at low protein concentrations (e.g. 5 mg/mL).36


According to the phase

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diagram that describes the protein crystallization behavior in a mixture of protein and precipitant at different concentrations,2 it will take a much long time for the sample to travel from the undersaturation zone through the metastable zone to the nucleation zone if the protein concentration is too low.

The protein concentration of 5 mg/mL is in this low

concentration regime. Figure 5 shows the percentages of the crystal growth, calculated by dividing the numbers of wells containing observable lysozyme crystals by the total numbers of wells used in the experiment.

It was found that both the 30-nm and 100-nm ND films

prepared at 50 and 500 µg/mL were able to yield substantially higher crystal formation rates than that of the controls on day 7 under the same experimental conditions.

Crystal structures.

To examine the quality of the protein crystals, we performed

X-ray diffraction to determine the structure of lysozyme.

Pairwise comparison using the

DALI server37 revealed that the structures of lysozyme in the protein crystals grown under the ND film conditions shared close similarities with that of the template (Table 1).

The

root-mean-square deviations are 0.6 Å (127 Cα atoms) and 0.7 Å (127 Cα atoms) for crystals grown in the presence of 30- and 100-nm ND films, respectively, suggesting that the use of ND as a nucleant does not pose a negative effect on the protein structure during the crystallization processes.

Both the crystals belong to the same space group P43212 and have

similar unit-cell parameters (a = 77.61, b = 77.61, c = 37.36 versus a = 77.11, b = 77.11, c = 37.07).

Interestingly, a higher resolution (1.41 Å) was achieved for crystals produced with

the 30-nm ND film method, compared to that (1.79 Å) of the 100-nm ND film method.

The

results support the notion that NDs can facilitate protein crystallization without compromising the quality (i.e. resolution) of structure determination.

DISCUSSION This work presents two forms of NDs as the nucleating agents for protein crystallization:


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aggregates and films.

To compare the protein crystallization efficiency between these two

methods, we grew lysozyme crystals under the same protein concentration of 20 mg/mL for 16 h.

As shown in Figure 6, more lysozyme crystals (size > 10 µm) could be observed in

the latter (c, d) than the former (a, b) cases, suggesting that the ND films can assist more effectively than the ND aggregates in protein nucleation and growth.

Since both the

nucleating agents are porous in structure, the result implies that the reduction of the tumbling motions of the protein crystals at initial stages of the growth makes significant contributions to these processes.

Additionally, the significantly larger size of the ND domains and

cavities in the deposited films than in the aggregates suspended in the solution can also contribute to the enhancement. The above experiments demonstrate that the presence of surface-oxidized NDs can indeed help reduce the threshold concentrations required for protein crystallization (Figure 5). Moreover, the ND films outperform the ND aggregates in such applications (Figure 6).

A

protein concentration in the range of 5 mg/mL is sufficient for the crystal growth of lysozyme with the aid of the ND films. RNase A (15 mg/mL). concentrations.

We have also applied this method to other proteins such as

However, no significant improvement was found at the specified

We attributed the disparity between these two results to the preferential

formation of protein clusters in the lysozyme solution.

These clusters, present in some

commercial lysozyme samples (such as the one used in this experiment),38 are likely to serve as inherent nucleation precursors in protein crystallization.39,40 Stimulated by the concept that protein clusters may play a key role in the crystallization process,38 we performed additional experiments using RNase A for testing.

We first

analyzed the sample solution (5 mg/mL) of the protein by DLS and then induced cluster formation by adding 3 M (NH4)2SO4 into the solution drop by drop while monitoring the sizes of the protein clusters formed in situ.

Figure 7a shows the hydrodynamic diameters of


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such formed RNase A clusters, which are typically 300 nm in size but have a broad distribution.

We employed these clusters in combination with 30-nm ND films as the

nucleating agents to facilitate the protein growth in the crystallization buffers.

Indeed, in

accord with the findings for lysozyme, the presence of pre-formed clusters significantly facilitated the protein crystallization processes.

Growth of RNase A crystals at the

concentration of 5 mg/mL was readily achieved with this protein cluster approach (Figure 7b – 7e).

Although only tens-micron-sized protein crystals were formed after 5 days of growth,

they are useful as microseeds for the optimization of protein crystallization by matrix screening.41,42 We note that the presently obtained result is consistent with the two-step mechanism of protein crystallization in solution, which proposes that protein clusters first form in a metastable intermediate phase, followed by nucleation of protein crystals within that phase.43,44

According to the mechanism, the addition of pre-formed protein clusters should

lower the energy barrier of the nucleation, as we have demonstrated in this work with two model proteins, lysozyme and RNase A.

The approach, comprising protein clusters and ND

films, could find useful applications in facilitating the crystallization of low abundant proteins under unfavorable growth conditions (such as the crystal growth at ultralow supersaturations36).

A challenge in future experiments is to apply the technique, in

combination with other nucleation methods (such as lipidic bicelles45,46 and laser trapping47), to assist membrane protein crystallization.

ACKNOWLEDGEMENTS This work was supported by Academia Sinica and the Ministry of Science and Technology, Taiwan, with Grant No. 104-2811-M-001-149.


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Table 1. Diffraction data collected from two lysozyme crystals grown in the presence of ND films made of 30 and 100 nm particles. Data collected

30 nm ND

100 nm ND

Space group

P43212

P43212

77.61, 77.61, 37.36

77.11, 77.11, 37.07

90, 90, 90

90, 90, 90

20-1.41 (1.46–1.41)

20-1.79 (1.85–1.79)

Unit cell

a, b, c (Å) α, β, γ (°) Resolution (Å)a a

Observed reflections

202096 (19863)

87377 (11695)

a

22553 (2207)

10677 (1093)

a

99.8 (100.0)

96.8 (99.8)

9.0 (9.0)

8.2 (10.7)

23.5 (3.6)

14.6 (5.9)

7.5 (49.9)

11.2 (41.0)

20-1.41 (1.45–1.41)

20-1.79 (1.86–1.79)

22138 (1388)

10598 (1085)

19.6 (20.6)

20.6 (23.9)

22.9 (27.0)

23.8 (28.7)

0.006

0.002

1.03

0.70

16.8

20.8

Favoured

97.6

96.8

Allowed

2.4

3.2

Disallowed

0

0

Unique reflections Completeness (%) a

Redundancy a

I/σ(I) Rmerge (%)a,b Refinement Resolution (Å)a a

Reflections (F > 0 σF) a,c

Rcryst (%)

a,d

Rfree (%) R.m.s. deviations Bond lengths (Å) Bond angles (°) 2

Mean B value (Å ) e

Ramachandran analysis (%)

a

Values in parentheses are for the highest resolution shells.

b

Rmerge ＝Σ|Ii – |/ΣIi, Ii is the average intensity value of the equivalent reflections.

c

Rcryst = Σ(|Fo – Fc|)/Σ|Fo|, Fo and Fc are observed and calculated structure factors, respectively.

d e

Rfree was calculated from 10% of data randomly excluded data from refinement.

The Ramachandran analysis was performed by MolProbity.


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Figure 1. Characterization of ND aggregates. Number-averaged size distributions of (a) 100 nm NDs and (b) 30 nm NDs in water and their aggregates in crystallization buffers. Insets: Corresponding optical microscopy images of ND aggregates (50 µg/mL) suspended in lysozyme buffers.

Scale bar: 500 µm.


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Figure 2. ND aggregates as nucleating agents.

Numbers of protein crystals formed in the

screening results with 98 buffers following three different treatments: control (black), 100 nm ND (red), and 30 nm ND (green).

The concentrations of 100 nm NDs and 30 nm NDs in the

buffers are both 50 µg/mL in 3 µL protein drops.


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Figure 3. Characterization of ND films.

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(a) Workflow of the ND film preparation (indicated

by arrow) using a drop of Piranha solution (red) to create a hydrophilic area on a sitting drop plate, followed by coating of the etched area with poly-L-lysine (green) and then with NDs (black).

(b, c) SEM images of ND films made of (b) 100 nm and (c) 30 nm particles.

Scale bars in (b) and (c) are 1 µm and 100 nm, respectively.

(d, e) Optical images of ND

films made of (d) 100 nm and (e) 30 nm particles with a concentration of 500 µg/mL on sitting drop plates.

Scale bar: 500 µm.


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Figure 4. ND films as nucleating agents.

The films are made of (a – d) 100 nm and (e – h)

30 nm NDs at the concentrations of (a, e) 0, (b, f) 50, (c, g) 250, and (d, h) 500 µg/mL. protein is lysozyme (20 mg/mL) and the crystal growth time is 16 h.


Scale bar: 500 µm.

The

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Figure 5. ND films as nucleating agents at low protein concentrations. The crystal growth ratio is defined as the number of wells having lysozyme crystal formation versus the total number of wells being tested. suspensions.

The ND films are prepared with either 50 or 500 µg/mL ND

The lysozyme concentration is 5 mg/mL, the crystal growth time is 160 h, and

control wells are not coated with NDs.


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Figure 6. Comparison of ND aggregates and ND films as nucleating agents.

Crystallization

of lysozyme (20 mg/mL) in the presence of (a, b) ND aggregates (a, 100 nm; b, 30 nm) and (c, d) ND films (c, 100 nm; d, 30 nm) prepared with a particle concentration of 500 µg/mL on sitting drop plates.

The crystal growth time is 16 h.

Scale bar: 500 µm.


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Figure 7. Protein clusters as nucleating agents.

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(a) Volume-averaged size distributions of

RNase A in water and their clusters after titration with 3 M (NH4)2SO4 solution.

(b – e)

Crystallization of RNase A (5 mg/mL) with or without 30-nm ND films in the presence or absence of pre-formed protein clusters.

(b) Control, (c) ND film only, (d) protein clusters

only, and (e) ND film and protein clusters.

The crystal growth time is 120 h.

500 µm.


Scale bar:

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Figure 1 82x131mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2 80x58mm (300 x 300 DPI)


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Figure 3 153x80mm (300 x 300 DPI)


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Figure 4 158x58mm (300 x 300 DPI)


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Figure 7 83x115mm (300 x 300 DPI)


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Table of Contents 156x103mm (300 x 300 DPI)


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Nanodiamonds as Nucleating Agents for Protein Crystallization

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