Recent advances in the microgravity crystallization of biological macromolecules

The status of microgravity research in the crystallization of biological macromolecules is presented. Currently, two paths of investigation are being undertaken. The first is the production of high-quality crystals in space for biotechnology and research applications. The second is the study of the mechanisms by which these superior crystals are formed in microgravity. Emphasis is also placed on macromolecules and the exploration of the flash-frozen-samples-Dewar approach for multiple crystallizations. Future space flight opportunities to continue this research are discussed.     

Protein and virus crystal growth on international microgravity laboratory-2.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.     

Physiology in microgravity.

Studies of physiology in microgravity are remarkably recent, with almost all the data being obtained in the past 40 years. The first human spaceflight did not take place until 1961. Physiological measurements in connection with the early flights were crude, but, in the past 10 years, an enormous amount of new information has been obtained from experiments on Spacelab. The United States and Soviet/Russian programs have pursued different routes. The US has mainly concentrated on relatively short flights but with highly sophisticated equipment such as is available in Spacelab. In contrast, the Soviet/Russian program concentrated on first the Salyut and then the Mir space stations. These had the advantage of providing information about long-term exposure to microgravity, but the degree of sophistication of the measurements in space was less. It is hoped that the International Space Station will combine the best of both approaches. The most important physiological changes caused by microgravity include bone demineralization, skeletal muscle atrophy, vestibular problems causing space motion sickness, cardiovascular problems resulting in postflight orthostatic intolerance, and reductions in plasma volume and red cell mass. Pulmonary function is greatly altered but apparently not seriously impaired. Space exploration is a new frontier with long-term missions to the moon and Mars not far away. Understanding the physiological changes caused by long-duration microgravity remains a daunting challenge.     

Protein crystallization and phase diagrams

The phase diagram is a map which represents the state of a material (e.g., solid and liquid) as a function of the ambient conditions (e.g., temperature and concentration). It is therefore a useful tool in processing many different classes of materials. In this article, methods to determine the phase diagram of an aqueous solution of a globular protein are described, focusing on the solid (crystal) and condensed liquid states. The use of the information contained in the phase diagram for protein crystallization is also discussed.     

Protein crystallization under high pressure

Pressure is expected to be an important parameter to control protein crystallization, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. So far, a lot of studies on protein crystallization have been done. Solubility of protein depends on pressure. For instance, the solubility of tetragonal lysozyme crystal increased with increasing pressure, while that of orthorhombic crystal decreased. The solubility of subtilisin increased with increasing pressure. Crystal growth rates of protein also depend on pressure. The growth rate of glucose isomerase was significantly enhanced with increasing pressure. The growth rate of tetragonal lysozyme crystal and subtilisin decreased with increasing pressure. To study the effects of pressure on the crystallization more precisely and systematically, hen egg white lysozyme is the most suitable protein at this stage, since a lot of data can be used. We focused on growth kinetics under high pressure, since extensive studies on growth kinetics have already been done at atmospheric pressure, and almost all of them have explained the growth mechanisms well. The growth rates of tetragonal lysozyme decreased with pressure under the same supersaturation. This means that the surface growth kinetics significantly depends on pressure. By analyzing the dependence of supersaturation on growth rate, it was found that the increase in average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in growth rate. At this stage, it is not clear whether the increase in surface energy with increasing pressure is the main reason or not. Fundamental studies on protein crystallization under high pressure will be useful for high pressure crystallography and high pressure protein science.     

Protein crystallization in the structural genomics era

There are five broad areas where noteworthy advances have occurred in the field of macromolecular crystallization in the past 10 years, though some areas have seen the major part of those advances in only the last two years. This is largely a consequence of the international structural genomics initiative and its early results. The five areas are: (1) Physical studies and characterization of the protein crystallization process; (2) Development of new practical approaches and procedures; (3) The implementation of protein engineering by genetic means to enhance both purification and crystallization; (4) The creation of new screening conditions based on information and databases emerging from structural genomics; and (5) Development and implementation of automation, robotics, and mass screening of crystallization conditions using very small amounts of protein. A brief summary is provided here of the progress in the past few years and the influence of the structural genomics project.     

Protein pattern of Xenopus laevis embryos grown in simulated microgravity

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.     

Ventilation-perfusion matching in long-term microgravity.

We studied the ventilation-perfusion matching pattern in normal gravity (1 G) and short- and long-duration microgravity (microG) using the cardiogenic oscillations in the sulfur hexaflouride (SF(6)) and CO(2) concentration signals during the phase III portion of vital capacity single-breath washout experiments. The signal power of the cardiogenic concentration variations was assessed by spectral analysis, and the phase angle between the oscillations of the two simultaneously expired gases was obtained through cross-correlation. For CO(2), a significant reduction of cardiogenic power was observed in microG, with respect to 1 G, but the reduction was smaller and more variable in the case of SF(6). A shift from an in-phase condition in 1 G to an out-of-phase condition was found for both short- and long-duration microG. We conclude that, although the distribution of ventilation and perfusion becomes more homogeneous in microG, significant inhomogeneities persist and that areas of high perfusion become associated with areas of relatively lower ventilation. In addition, these modifications seem to remain constant during long-term exposure to microG.     

Protein crystallization: virtual screening and optimization

Advances in genomics have yielded entire genetic sequences for a variety of prokaryotic and eukaryotic organisms. This accumulating information has escalated the demands for three-dimensional protein structure determinations. As a result, high-throughput structural genomics has become a major international research focus. This effort has already led to several significant improvements in X-ray crystallographic and nuclear magnetic resonance methodologies. Crystallography is currently the major contributor to three-dimensional protein structure information. However, the production of soluble, purified protein and diffraction-quality crystals are clearly the major roadblocks preventing the realization of high-throughput structure determination.

This paper discusses a novel approach that may improve the efficiency and success rate for protein crystallization. An automated nanodispensing system is used to rapidly prepare crystallization conditions using minimal sample. Proteins are subjected to an incomplete factorial screen (balanced parameter screen), thereby efficiently searching the entire “crystallization space” for suitable conditions. The screen conditions and scored experimental results are subsequently analyzed using a neural network algorithm to predict new conditions likely to yield improved crystals. Results based on a small number of proteins suggest that the combination of a balanced incomplete factorial screen and neural network analysis may provide an efficient method for producing diffraction-quality protein crystals.     

Protein phase separation and determinants of in cell crystallization

Liquid‐liquid phase separation (LLPS) in cells is known as a complex physicochemical process causing the formation of membrane‐less organelles (MLOs). Cells have well‐defined different membrane‐surrounded organelles like mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, etc., however, on demand they can create MLOs as stress granules, nucleoli and P bodies to cover vital functions and regulatory activities. However, the mechanism of intracellular molecule assembly into functional compartments within a living cell remains till now not fully understood. in vitro and in vivo investigations unveiled that MLOs emerge after preceding liquid‐liquid, liquid‐gel, liquid‐semi‐crystalline, or liquid‐crystalline phase separations. Liquid‐liquid and liquid‐gel MLOs form the majority of cellular phase separation events, while the occurrence of micro‐sized crystals in cells was only rarely observed, however can be considered as a result of a preceding protein phase separation event. In vivo, also known and termed as in cellulo crystals, are reported since 1853. In some cases, they have been linked to vital cellular functions, such as storage and detoxification. However, the occurrence of in cellulo crystals is also associated to diseases like cataract, hemoglobin C diseases, etc. Therefore, better knowledge about the involved molecular processes will support drug discovery investigations to cure diseases related to in cellulo crystallization. We summarize physical and chemical determinants known today required for phase separation initiation and formation and in cellulo crystal growth. In recent years it has been demonstrated that LLPS plays a crucial role in cell compartmentalization and formation of MLOs. Here we discuss potential mechanisms and potential crowding agents involved in protein phase separation and in cellulo crystallization.     

Cerebral hemodynamics during microgravity.

As one of the causes of the space adaptation syndrome, an increased intracranial pressure due to the cephalad fluid shift is suggested. In the present study, we measured intracranial pressure (ICP), aortic pressure and cerebral flow velocity (CFV) in anesthetized rats (n=5) during 4.5 sec of microgravity induced by free drop. The rats were set at horizontal prone (Flat) and 30-degree head-up whole body tilting (HU) positions to examine the effect of gravitational pressure gradient. Then, arterial pressure at the eye level (APeye), cerebral perfusion pressure (CPP; CPP=APeye-ICP), and CPP-CFV relationship was calculated. In HU position, ICP, APeye, and CPP increased by 2.2 +/- 0.4, 12.3 +/- 2.0, and 10.1 +/- 1.7 mmHg respectively. However, CFV did not change significantly. In Flat position, none of these variables did not change significantly. In HU position the slope of CPP-CFV relationship decreased, suggesting the increased cerebral flow resistance. However, it did not change in Flat position. These results can be understood by the disappearance of gravitational pressure gradient by microgravity and the cerebral autoregulation.     

Embryonic quail eye development in microgravity.

The US-Russian joint quail embryo project was designed to study the effects of microgravity on development of Japanese quail embryos incubated aboard Mir. For this part of the project, eyes from embryonic days 14 and 16 (E14 and E16) flight embryos were compared with eyes from several groups of ground-based control embryos. Measurements were recorded for eye weights; eye, corneal, and scleral ring diameters; and numbers of bones in scleral ossicle rings. Transparency of E16 corneas was documented, and immunohistochemical staining was performed to observe corneal innervation. In addition, corneal ultrastructure was observed at the electron microscopic level. Except for corneal diameter of E16 flight embryos, compared with that of one of the sets of controls, results reported here indicate that eye development occurred normally in microgravity. Fixation by cracking the shell and placing the egg in paraformaldehyde solution did not adequately preserve corneal nerves or cellular ultrastructure.     

Protein crystallization by design: chymotrypsinogen without precipitants

Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.     

Protein crystallization for genomics: throughput versus output

Structural genomics involves many steps in order to reach from Gene to structure. This article focuses on the crystallization step in this chain of tasks. It is becoming increasingly evident that the current high throughput procedures for crystallising proteins do not always produce the expected output of high quality crystals required for structure determination by x-ray crystallography. This problem is discussed and suggestions for raising the output are presented.     

Protein crystallization analysis on the World Community Grid

We have developed an image-analysis and classification system for automatically scoring images from high-throughput protein crystallization trials. Image analysis for this system is performed by the Help Conquer Cancer (HCC) project on the World Community Grid. HCC calculates 12,375 distinct image features on microbatch-under-oil images from the Hauptman-Woodward Medical Research Institute’s High-Throughput Screening Laboratory. Using HCC-computed image features and a massive training set of 165,351 hand-scored images, we have trained multiple Random Forest classifiers that accurately recognize multiple crystallization outcomes, including crystals, clear drops, precipitate, and others. The system successfully recognizes 80% of crystal-bearing images, 89% of precipitate images, and 98% of clear drops.     

Physiology of a microgravity environment invited review: microgravity and skeletal muscle.

Spaceflight (SF) has been shown to cause skeletal muscle atrophy; a loss in force and power; and, in the first few weeks, a preferential atrophy of extensors over flexors. The atrophy primarily results from a reduced protein synthesis that is likely triggered by the removal of the antigravity load. Contractile proteins are lost out of proportion to other cellular proteins, and the actin thin filament is lost disproportionately to the myosin thick filament. The decline in contractile protein explains the decrease in force per cross-sectional area, whereas the thin-filament loss may explain the observed postflight increase in the maximal velocity of shortening in the type I and IIa fiber types. Importantly, the microgravity-induced decline in peak power is partially offset by the increased fiber velocity. Muscle velocity is further increased by the microgravity-induced expression of fast-type myosin isozymes in slow fibers (hybrid I/II fibers) and by the increased expression of fast type II fiber types. SF increases the susceptibility of skeletal muscle to damage, with the actual damage elicited during postflight reloading. Evidence in rats indicates that SF increases fatigability and reduces the capacity for fat oxidation in skeletal muscles. Future studies will be required to establish the cellular and molecular mechanisms of the SF-induced muscle atrophy and functional loss and to develop effective exercise countermeasures.