The Study of the Mechanism of Protein Crystallization in Space by Using Microchannel to Simulate Microgravity Environment

Space is expected to be a convection-free, quiescent environment for the production of large-size and high-quality protein crystals. However, the mechanisms by which the diffusion environment in space improves the quality of the protein crystals are not fully understood. The interior of a microfluidic device can be used to simulate a microgravity environment to investigate the protein crystallization mechanism that occurs in space. In the present study, lysozyme crystals were grown in a prototype microchannel device with a height of 50 μm in a glass-polydimethylsiloxane (PDMS)-glass sandwich structure. Comparative experiments were also conducted in a sample pool with a height of 2 mm under the same growth conditions. We compared the crystal morphologies and growth rates of the grown crystals in the two sample pools. The experimental results showed that at very low initial supersaturation, the morphology and growth rates of lysozyme crystals under the simulated microgravity conditions is similar to that on Earth. With increasing initial supersaturation, a convection-free, quiescent environment is better for lysozyme crystal growth. When the initial supersaturation exceeded a threshold, the growth of the lysozyme crystal surface under the simulated microgravity conditions never completely transform from isotropic to anisotropic. The experimental results showed that the convection may have a dual effect on the crystal morphology. Convection can increase the roughness of the crystal surface and promote the transformation of the crystal form from circular to tetragonal during the crystallization process.

Download Full-text

Methods for Obtaining Better Diffractive Protein Crystals: From Sample Evaluation to Space Crystallization

Crystals ◽

10.3390/cryst10020078 ◽

2020 ◽

Vol 10 (2) ◽

pp. 78

Author(s):

Yoshinobu Hashizume ◽

Koji Inaka ◽

Naoki Furubayashi ◽

Masayuki Kamo ◽

Sachiko Takahashi ◽

...

Keyword(s):

Crystal Growth ◽

Quality Evaluation ◽

Protein Crystallization ◽

Protein Crystals ◽

Microgravity Environment ◽

Theoretical Understanding ◽

Protein Sample ◽

Space Experiments ◽

Flash Cooling ◽

Crystallization Conditions

In this paper, we present a summary on how to obtain protein crystals from which better diffraction images can be produced. In particular, we describe, in detail, quality evaluation of the protein sample, the crystallization conditions and methods, flash-cooling protection of the crystal, and crystallization under a microgravity environment. Our approach to protein crystallization relies on a theoretical understanding of the mechanisms of crystal growth. They are useful not only for space experiments, but also for crystallization in the laboratory.

Download Full-text

Highly Purified Glucose Isomerase Crystals Under Microgravity Conditions Grow as Fast as Those on the Ground Do

10.26434/chemrxiv.11808504.v1 ◽

2020 ◽

Author(s):

Yoshihisa Suzuki

Keyword(s):

Growth Rates ◽

Glucose Isomerase ◽

Spiral Growth ◽

Protein Crystals ◽

In Situ Observation ◽

Lateral Growth ◽

Impurity Incorporation ◽

Microgravity Conditions

Suppression of convection flows (solute transportation) and that of impurity incorporation into crystals seem to be the main reasons why the quality of protein crystals becomes better under microgravity conditions, whereas each precise mechanism has not been completely clarified yet. We tried to clarify the former reason by the in-situ observation of spiral growth hillocks on the {110} faces of highly purified glucose isomerase (GI) crystals under microgravity conditions and on the ground. Lateral growth rates Vlateral of a spiral hillock on the {110} face of a glucose isomerase crystal in situ under microgravity conditions and step velocities Vstep of the same configuration on the ground took similar values as far as the maximum values are compared each other. This similarity indicates there are less influences of the convection flows on the growth rates of protein crystals contrary to conventional expectations.

Download Full-text

Highly Purified Glucose Isomerase Crystals Under Microgravity Conditions Grow as Fast as Those on the Ground Do

10.26434/chemrxiv.11808504 ◽

2020 ◽

Author(s):

Yoshihisa Suzuki

Keyword(s):

Growth Rates ◽

Glucose Isomerase ◽

Spiral Growth ◽

Protein Crystals ◽

In Situ Observation ◽

Lateral Growth ◽

Impurity Incorporation ◽

Microgravity Conditions

Download Full-text

A magnetically enabled simulation of microgravity represses the auxin response during early seed germination on a microfluidic platform

Microsystems & Nanoengineering ◽

10.1038/s41378-021-00331-5 ◽

2022 ◽

Vol 8 (1) ◽

Author(s):

Jing Du ◽

Lin Zeng ◽

Zitong Yu ◽

Sihui Chen ◽

Xi Chen ◽

...

Keyword(s):

Simulated Microgravity ◽

Critical Factor ◽

Vital Role ◽

Microgravity Environment ◽

Biological Study ◽

Gravitational Acceleration ◽

Auxin Response ◽

Microfluidic Platform ◽

Microgravity Conditions ◽

On Chip

AbstractFor plants on Earth, the phytohormone auxin is essential for gravitropism-regulated seedling establishment and plant growth. However, little is known about auxin responses under microgravity conditions due to the lack of a tool that can provide an alteration of gravity. In this paper, a microfluidic negative magnetophoretic platform is developed to levitate Arabidopsis seeds in an equilibrium plane where the applied magnetic force compensates for gravitational acceleration. With the benefit of the microfluidic platform to simulate a microgravity environment on-chip, it is found that the auxin response is significantly repressed in levitated seeds. Simulated microgravity statistically interrupts auxin responses in embryos, even after chemical-mediated auxin alterations, illustrating that auxin is a critical factor that mediates the plant response to gravity alteration. Furthermore, pretreatment with an auxin transportation inhibitor (N-1-naphthylphthalamic acid) enables a decrease in the auxin response, which is no longer affected by simulated microgravity, demonstrating that polar auxin transportation plays a vital role in gravity-regulated auxin responses. The presented microfluidic platform provides simulated microgravity conditions in an easy-to-implement manner, helping to study and elucidate how plants correspond to diverse gravity conditions; in the future, this may be developed into a versatile tool for biological study on a variety of samples.

Download Full-text

The nucleation of protein crystals as a race against time with on- and off-pathways

Journal of Applied Crystallography ◽

10.1107/s1600576717007312 ◽

2017 ◽

Vol 50 (4) ◽

pp. 1056-1065 ◽

Cited By ~ 6

Author(s):

Cecilia Ferreira ◽

Silvia Barbosa ◽

Pablo Taboada ◽

Fernando A. Rocha ◽

Ana M. Damas ◽

...

Keyword(s):

Hydrodynamic Radius ◽

Protein Crystallization ◽

Initial Step ◽

Protein Crystals ◽

Protein Oligomerization ◽

High Supersaturation ◽

Well Differentiated ◽

On And Off Pathways ◽

The Right ◽

Lysozyme Crystals

High supersaturation levels are a necessary but insufficient condition for the crystallization of purified proteins. Unlike most small molecules, proteins can take diverse aggregation pathways that make the outcome of crystallization assays quite unpredictable. Here, dynamic light scattering and optical microscopy were used to show that the nucleation of lysozyme crystals is preceded by an initial step of protein oligomerization and by the progressive formation of metastable clusters. Because these steps deplete the concentration of soluble monomers, the probability of obtaining protein crystals decreases as time progresses. Stochastic variations of the induction time are thus amplified to a point where fast crystallization can coexist with unyielding regimes in the same conditions. With an initial hydrodynamic radius of ∼100 nm, the metastable clusters also promote the formation of protein crystals through a mechanism of heterogeneous nucleation. Crystal growth (on-pathway) takes place in parallel with cluster growth (off-pathway). The Janus-faced influence of the mesoscopic clusters is beneficial when it accelerates the formation of the first precrystalline nuclei and is detrimental as it depletes the solution of protein ready to crystallize. Choosing the right balance between the two effects is critical for determining the success of protein crystallization trials. The results presented here suggest that a mild oligomerization degree promotes the formation of a small number of metastable clusters which then catalyze the nucleation of well differentiated crystals.

Download Full-text

Bridgman Crystal Growth of an Alloy With Thermosolutal Convection Under Microgravity Conditions

Journal of Heat Transfer ◽

10.1115/1.1389058 ◽

2001 ◽

Vol 123 (5) ◽

pp. 990-998 ◽

Cited By ~ 5

Author(s):

James E. Simpson ◽

Suresh V. Garimella ◽

Henry C. de Groh ◽

Reza Abbaschian

Keyword(s):

Crystal Growth ◽

Growth Conditions ◽

Convective Cell ◽

Two Dimensions ◽

Experimental Results ◽

Thermosolutal Convection ◽

Microgravity Environment ◽

Uniform Grid ◽

Microgravity Conditions ◽

Bridgman Crystal Growth

The solidification of a dilute alloy (bismuth-tin) under Bridgman crystal growth conditions is investigated. Computations are performed in two dimensions with a uniform grid. The simulation includes the species concentration, temperature and flow fields, as well as conduction in the ampoule. Fully transient simulations have been performed, with no simplifying steady state approximations. Results are obtained under microgravity conditions for pure bismuth, and for Bi-0.1 at.% Sn and Bi-1.0 at.% Sn alloys, and compared with experimental results obtained from crystals grown in the microgravity environment of space. For the Bi-1.0 at.% Sn case the results indicate that a secondary convective cell, driven by solutal gradients, forms near the interface. The magnitude of the velocities in this cell increases with time, causing increasing solute segregation at the solid/liquid interface. Finally, a comparison between model predictions and results obtained from a space experiment is reported. The concentration-dependence of the alloy melting temperature is incorporated in the model for this case. Satisfactory correspondence is obtained between the predicted and experimental results in terms of solute concentrations in the solidified crystal.

Download Full-text

SOLUBILITY MEASUREMENTS OF TETRAGONAL LYSOZYME CRYSTALS

International Journal of Modern Physics B ◽

10.1142/s0217979206040957 ◽

2006 ◽

Vol 20 (25n27) ◽

pp. 4117-4122 ◽

Cited By ~ 2

Author(s):

YOSHIHISA SUZUKI ◽

ATSUTO ARAI ◽

KATSUHIRO TAMURA

Keyword(s):

Growth Rates ◽

The Difference ◽

Lysozyme Crystals ◽

Lysozyme Crystal

Solubility of tetragonal lysozyme crystal was measured by using two different methods. The solubility measured by using growth rates of the crystal corresponded well to that measured by using change in the concentration of the supernatant of the solution. Our data also corresponded well to the data obtained by Sazaki et al. and Gray et al., while not to those obtained by Howard et al. and Rosenberger et al. The discrepancies are due to the difference in the principles of the methods. We also checked effects of different initial concentrations on the solubility. Contrary to previous reports, the solutions of the different initial concentrations did not reach equilibrium when their concentrations attained to a same value.

Download Full-text

Alterations in the Virulence Potential of Enteric Pathogens and Bacterial–Host Cell Interactions Under Simulated Microgravity Conditions

Journal of Toxicology and Environmental Health Part A ◽

10.1080/15287390500361792 ◽

2006 ◽

Vol 69 (14) ◽

pp. 1345-1370 ◽

Cited By ~ 53

Author(s):

V. Chopra ◽

A. A. Fadl ◽

J. Sha ◽

S. Chopra ◽

C. L. Galindo ◽

...

Keyword(s):

Host Cell ◽

Simulated Microgravity ◽

Cell Interactions ◽

Enteric Pathogens ◽

Bacterial Host ◽

Virulence Potential ◽

Host Cell Interactions ◽

Microgravity Conditions

Download Full-text

The Effects of Simulated Microgravity on Macrophage Phenotype

Biomedicines ◽

10.3390/biomedicines9091205 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1205

Author(s):

Christopher Ludtka ◽

Erika Moore ◽

Josephine B. Allen

Keyword(s):

Protein Expression ◽

Simulated Microgravity ◽

Prolonged Exposure ◽

Cell Function ◽

Immune Cell ◽

Expression Profiles ◽

Microgravity Environment ◽

M2 Macrophage ◽

Macrophage Function ◽

Gene And Protein Expression

The effects of spaceflight, including prolonged exposure to microgravity, can have significant effects on the immune system and human health. Altered immune cell function can lead to adverse health events, though precisely how and to what extent a microgravity environment impacts these cells remains uncertain. Macrophages, a key immune cell, effect the inflammatory response as well as tissue remodeling and repair. Specifically, macrophage function can be dictated by phenotype that can exist between spectrums of M0 macrophage: the classically activated, pro-inflammatory M1, and the alternatively activated, pro-healing M2 phenotypes. This work assesses the effects of simulated microgravity via clinorotation on M0, M1, and M2 macrophage phenotypes. We focus on phenotypic, inflammatory, and angiogenic gene and protein expression. Our results show that across all three phenotypes, microgravity results in a decrease in TNF-α expression and an increase in IL-12 and VEGF expression. IL-10 was also significantly increased in M1 and M2, but not M0 macrophages. The phenotypic cytokine expression profiles observed may be related to specific gravisensitive signal transduction pathways previously implicated in microgravity regulation of macrophage gene and protein expression. Our results highlight the far-reaching effects that simulated microgravity has on macrophage function and provides insight into macrophage phenotypic function in microgravity.

Download Full-text