空间微重力环境下蛋白质晶体生长的研究进展

空间微重力环境可消除或减弱常重力场下溶液中存在的对流和沉降,为蛋白质晶体生长提供一个相对均一和稳定的环境,有利于得到尺寸更大、衍射分辨率更高的蛋白质晶体。通过对这些高质量空间晶体进行X射线衍射分析,可获得多种蛋白质的精细三维结构。从空间蛋白质晶体生长的发展历史、研究成果、生长机理、存在的问题与对策等方面总结了空间微重力环境下蛋白质晶体生长的研究进展,展望了空间蛋白质结晶的未来。     

Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.     

The space experiment of protein crystallization aboard the Chinese spacecraft SZ-3

Using new flight hardware, a Chinese mission of space protein crystallization has been performed aboard the Chinese spacecraft SZ-3. Preliminary analyses of the experimental results have shown that a few proteins produced better crystals in space. At least, the crystals of cytochrome b5 mutant could diffract X-ray beyond the highest resolution reported so far for the same kind of crystals. In addition, some rules derived from our numerical studies of the liquid/liquid diffusion protein crystallization were proved by the crystallization of lysozyme as model protein in this space experiment, which also clearly showed the advantages and disadvantages of the gelation of the protein solution used in microgravity growth of protein crystals.     

用于晶体生长的地基无容器悬浮技术

空间微重力环境下几乎无对流和沉降,可为晶体生长提供一个相对稳定和均一的理想环境,易于得到尺寸较大的高质量单晶。但是,空间结晶实验成功率低,费用昂贵,实验机会受限。因此,研发各种空间微重力环境地基模拟技术具有重要意义。目前可用于晶体生长的地基无容器悬浮技术主要有空气动力悬浮、静电悬浮、电磁悬浮、液体界面悬浮、超声悬浮和磁场悬浮技术等。这些地基模拟技术可实现晶体的无容器悬浮生长,避免器壁对晶体生长的不良影响,提高晶体质量,为解决X射线单晶衍射技术中的瓶颈问题提供新途径,还可为在地基进行结晶动力学和机理研究提供简单易行的方法。从技术原理、优势、缺陷及在结晶(特别是蛋白质结晶)中的应用4个方面对这些技术逐一进行了介绍和评述。重点介绍了液体界面悬浮、超声悬浮和磁场悬浮技术这3种用于蛋白质晶体生长的较为成熟的地基无容器悬浮技术。     

Protein crystallization in space

The microgravity environment of space is an ideal place to study the complicated protein crystallization process and to grow good-quality protein crystals. A series of crystal growth experiments of 10 different proteins was carried out in space on a Chinese re-entry satellite FSW-2 in August, 1992. The experiments were performed for about two weeks at a temperature of 18.5 +/- 0.5 degrees C using a tube-like crystallization apparatus made in the Shanghai Institute of Technical Physics, Academia Sinica. More than half of 48 samples from 6 proteins produced crystals, and the effects of microgravity on protein crystal growth were observed, especially for hen-egg white lysozyme and an acidic phospholipase A2 from the venom of Agkistrodon halys Pallas. Analyses of the crystallization of these two enzymes in this mission showed that the microgravity environment in space may be beneficial to improve size, external perfection, morphology, internal order, and nucleation of protein crystals. Some of these positive microgravity effects were also demonstrated by the growth of protein crystals in gelled solution with the above two enzymes. A structural analysis of the tetragonal lysozyme crystal grown in space is in progress.     

Effects of a microgravity environment on the crystallization of biological macromolecules

Macromolecules crystals are indispensable intermediates in the analysis of macromolecular structure, are essential for the application of x-ray diffraction methods, and are at the same time the greatest obstacle to success. Protein crystals are generally difficult to grow, often of imperfect form or small size, and frequently lack sufficient order. Their growth has become the rate limiting step in x-ray crystallography. Evidence has emerged from protein crystallization experiments carried out in space that suggests macromolecular crystals of improved order and quality can be grown in a microgravity environment. Presumably the absence of density driven convection and sedimentation permits a more deliberate and graceful entry of individual molecules into the crystal lattice. This in turn results in improvements in both morphology and the diffraction patterns of the crystals. The precise mechanisms for these improvements and the means for their optimization are, however, not understood at more than a rudimentary level. I attempt here to review microgravity effects that may play a role in protein crystal growth, sedimentation, convection and surface contact, and suggest their possible mechanisms.     

Colloidal hard-sphere crystallization kinetics in microgravity and normal gravity

The hard-sphere disorder-order transition serves as the paradigm for crystallization. We used time-resolved Bragg light scattering from the close-packed planes to measure the kinetics of nucleation and growth of colloidal hard-sphere crystals. The effects of gravity are revealed by comparison of the experiments in microgravity and normal gravity. Crystallites grow faster and larger in microgravity, and the coarsening between crystallites is suppressed by gravity. The face-centered-cubic structure was strongly indicated as being the stable structure for hard-sphere crystals. For a sample with a volume fraction of 0.552, the classic nucleation and growth picture is followed.     

High-throughput screens for postgenomics: studies of protein crystallization using microsystems technology

This paper describes the fabrication of a micromachined miniaturized array of chambers in a 2-mm-thick single crystal (100) silicon substrate for the combinatorial screening of the conditions required for protein crystallization screening (including both temperature and the concentration of crystallization agent). The device was fabricated using standard photolithography techniques, reactive ion etching (RIE) and anisotropic silicon wet etching to produce an array of 10 x 10 microchambers, with each element having a volume of 5 microL. A custom-built temperature controller was used to drive two peltier elements in order to maintain a temperature gradient (between 12 and 40 degrees C) across the device. The performance of the microsystem was illustrated by studying the crystallization of a model protein, hen egg white lysozyme. The crystals obtained were studied using X-ray diffraction at room temperature and exhibited 1.78 A resolution. The problems of delivering a robust crystallization protocol, including issues of device fabrication, delivery of a reproducible temperature gradient, and overcoming evaporation are described.     

The Protein Crystallisation Diagnostics Facility (PCDF) on Board ESA Columbus Laboratory

Crystals of proteins or macromolecules are at the basis of X-ray crystallography to reveal structural information necessary for the understanding of their likely mode of action. However, the structural resolution is strongly dependent on the crystalline quality, which is known to be related to gravity dependent processes. The facilities and instruments used so far to grow crystals in space have mostly focused on the growing of crystals for detailed post-flight analysis on ground, and less on the understanding of phenomena associated to the crystallisation processes. The Protein Crystallisation Diagnostics Facility (PCDF), developed by Astrium under contract of the European Space Agency (ESA), allows to study with several diagnostics means in situ the crystallisation of macromolecules over long periods in microgravity. In addition, several ground models with PCDF similar capabilities were developed to allow scientists to prepare their experiments. The PCDF is installed in the European Drawer Rack (EDR), on board ESA’s Columbus Laboratory module launched in February 2008 to the International Space Station (ISS) for research in microgravity on protein nucleation and assembling sequences. The PCDF configuration for this first mission accommodates four reactors, using the batch crystallization technique. Individual process control for temperature and concentration will allow several crystallizations of solutions to be performed. Each reactor will be observed by several optical diagnostics, including video microscopy, dynamic light scattering, and Mach–Zehnder interferometry. This paper presents the overall PCDF design and details the different diagnostics allowing the scientific community to use the PCDF in orbit for microgravity research on molecule assemblies grown from solutions.     

Effect of convective disturbances induced by g-jitter on the periodic precipitation of lysozyme

Numerical simulations are carried out to investigate the crystallization process of a protein macromolecular substance under two different conditions: pure diffusive regime and microgravity conditions present on space laboratories. The configuration under investigation consists of a protein reactor and a salt chamber separated by an “interface”. The interface is strictly related to the presence of agarose gel in one of the two chambers. Sedimentation and convection under normal gravity conditions are prevented by the use of gel in the protein chamber (pure diffusive regime). Under microgravity conditions periodic time-dependent accelerations (g-jitter) are taken into account. Novel mathematical models are introduced to simulate the complex phenomena related to protein nucleation and further precipitation (or resolution) according to the concentration distribution and in particular to simulate the motion of the crystals due to g-jitter in the microgravity environment. The numerical results show that gellified lysozyme (crystals “locked” on the matrix of agarose gel) precipitates to produce “spaced deposits”. The crystal formation results modulated in time and in space (Liesegang patterns), due to the non-linear interplay among transport, crystal nucleation and growth. The propagation of the nucleation front is characterized by a wavelike behaviour. In microgravity conditions (without gel), g-jitter effects act modifying the phenomena with respect to the on ground gellified configuration. The role played by the direction of the applied sinusoidal acceleration with respect to the imposed concentration gradient (parallel or perpendicular) is investigated. It has a strong influence on the dynamic behaviour of the depletion zones and on the spatial distribution of the crystals. Accordingly the possibility to obtain better crystals for diffraction analyses is discussed.     

用于蛋白质结晶的形核剂研究综述

获取蛋白质晶体是蛋白质三维结构解析、医疗药品生产、自组装纳米体系构建等过程中重要的步骤。例如,利用X射线衍射技术对蛋白质进行三维结构解析时,首先需要通过结晶条件筛选,获得质量较高的蛋白质晶体,进而进行衍射得到蛋白质结构相关信息。蛋白质结晶需要经历从未饱和区经亚稳区至形核区的形核过程以及从形核区到亚稳区的生长成熟过程。在整个蛋白质结晶过程中,形核过程是至关重要的一步。均相形核过程中,结晶体系中各个部分形核概率相同,当蛋白质结晶体系中溶液的过饱和度足够克服形核势垒时,在形核区发生成核,因而在低浓度的结晶溶液体系中,均相形核存在一定的局限性。形核剂的添加使蛋白质晶体异相形核,相较于均相形核其需要克服的阻力小,形核势垒低。因而形核剂的使用对于难结晶蛋白或者起始浓度过低的蛋白质结晶具有重要意义。随着结构生物学的发展,形核剂在蛋白质结晶中的研究仍是结晶方法学领域的热点问题。多孔微球对蛋白质分子的吸附作用有利于无序蛋白质分子团簇的形成,进而促进蛋白质形核。添加多孔微球不但可以增加结晶条件筛选数,也可以提高晶体质量。促进蛋白质分子有序排列的形核剂籽晶的使用,使晶体的形核生长过程始终处于结晶体系溶液浓度较低的状态,而交联的籽晶因为稳定性更高而更有应用前景。新型交互扩散结晶板中,蛋白质结晶体系通过一个较缓慢的交互扩散过程实现蛋白质结晶溶液浓度的变化,并且结晶体系可达到共平衡,因而能显著提高蛋白质晶体结晶条件筛选数和晶体质量:蛋白酶K结晶条件数由39个提升至47个,分辨率由1.66提升至1.54。利用基底材料的一些特性,如静电作用、疏水作用和氢键,可以起到促进蛋白质分子聚集的功能,从而促进形核。本文从物理作用和化学作用两个角度详细总结了形核剂对蛋白质结晶的影响,并展望了该领域的发展前景及研究方向。     

A Compact Device for Colloidal Crystal Studies on Tiangong-1 Target Spacecraft

An experimental device with three crystallization cells, each with two working positions, was designed to study growth kinetics and structural transformation of colloidal crystals under microgravity condition. The device is capable of remote control of experimental procedures. It uses direct-space imaging with white light to monitor morphology of the crystals and reciprocal-space laser diffraction (Kossel lines) to reveal lattice structure. The device, intended for colloidal crystal growth kinetics and structural transformation on Tiangong-1 target spacecraft, had run on-orbit for more than one year till the end of the mission. Hundreds of images and diffraction patterns were collected via the on-ground data receiving station. The data showed that single crystalline samples were successfully grown on the orbit. Structural transformation was carefully studied under electric and thermal field. Using a backup device, control experiments were also performed on the ground under similar conditions except for the microgravity. Preliminary results indicated that the on-orbit crystals were more stable than the on-ground ones.     

A New Paradigm of Crystallization Arising from Non-standard Nucleation Pathways

There is increasing evidence that large classes of colloid materials crystallize via a non-standard nucleation mechanism involving intermediate metastable phases. In this paper recent work on the microscopic derivation of the phase diagram and free energy barriers in the nucleation of protein crystals, and on the kinetics of growth of solid particles in the post-nucleation regime is reviewed. The extent to which combined structural and density fluctuation give rise to favourable crystallization pathways involving an intermediate fluid phase is assessed and the connection with experiments in microgravity at ISS (PROMISS-2 and NANOSLAB-2) is discussed.     

Effect of microgravity on crystallization in heavy metal fluoride glasses processed on the CSAR-I sounding rocket

Gravity-driven density segregation in viscous glass is believed to trigger homogeneous nucleation during the high-temperature processing of heavy metal fluoride (HMF) glasses. Processing of HMF glasses in microgravity could, therefore, minimize commonly observed micro-crystal formation in these glasses during their heat treatment for fibre drawing. Although, preliminary experiments on parabolic flight aircraft had earlier indicated that gravity enhances and microgravity suppresses crystallization during the processing of HMF glasses, these results were considered inconclusive due to the short processing time of 20 seconds. The CSAR-I sounding rocket provided an opportunity to process HMF glasses over a longer duration of five minutes in microgravity. These experiments indicated that microgravity helps in reducing crystallization in HMF glasses during their heat treatment at 325°C, which is very close to their fibre drawing temperature range of 300–320°C.     

Effect of rotation on surface tension driven flow during protein crystallization

The effect of rotation on surface tension gradient driven flow, also known as Marangoni convective flow, during protein crystallization is modeled and studied computationally under microgravity conditions, where the surface tension gradient force is the main significant driving force. The main parameters are the solutal Marangoni number M_c, representing the surface tension gradient force and the Taylor number Ta representing the rotational effect. The numerical computations for various values of the parameters and low gravity levels indicated nontrivial competing effects, due to surface tension gradient, centrifugal and Coriolis forces on the flow adjacent to the protein crystal interface and the associated solute flux. In particular, for given values of M_c, certain values of Ta were detected where the Sherwood number (Sh), representing the convective solute flux, and the convective flow effects are noticeably reduced. These results can provide conditions under which convective flow transport during the protein crystallization approaches the diffusion limited transport, which is desirable for the production of higher quality protein crystals.     

Effect of method of crystallization on the IV-III and IV-II polymorphic transitions of ammonium nitrate

A study has been undertaken on the effect of crystallization method on the IV<-->III transition of ammonium nitrate (AN). AN is crystallized in three different ways, viz. recrystallization, evaporative crystallization and melt crystallization. When the samples were crystallized from saturated aqueous solution, ideal crystals were formed, which behaved differently from the crystals formed from the other methods. The DTA examination of the crystals showed that the crystals have different transition behaviour. The moisture uptake of the samples determined were found to have influenced by the mode of crystallization. The samples were further analyzed by powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). The present study showed that the parameters like thermal history, number of previous transformations and moisture content have a very negligible influence on the IV<-->III transition of AN as compared to the method of crystallization.     

Counterdiffusion protein crystallisation in microgravity and its observation with PromISS (protein microscope for the international space station)

The crystallisation by counterdiffusion is a very efficient technique for obtaining high-quality protein crystals. A prerequisite for the use of counterdiffusion techniques is that mass transport must be controlled by diffusion alone. Sedimentation and convection can be avoided by either working in gelled systems, working in systems of small dimensions, or in the absence of gravity. We present the results from experiments performed on the ISS using the Protein Microscope for the International Space Station (PromISS), using digital holography to visualise crystal growth processes. We extensively characterised three model proteins for these experiments (cablys3*lysozyme, triose phosphate isomerase, and parvalbumin) and used these to assess the ISS as an environment for crystallisation by counterdiffusion. The possibility to visualise growth and movement of crystals in different types of experiments (capillary counterdiffusion and batch-type) is important, as movement of crystals is clearly not negligible.     

Protein crystal growth in microgravity using a liquid/liquid diffusion method

Crystallization of proteins by liquid liquid diffusion method was performed in microgravity using the MDA Minilab aboard the US Space Shuttle. Three proteins, namely lysozyme, trichosanthin, and a new lechin, were crystallized in the space experiment. In contrast to the results of space experiments with a tube-like vapor diffusion method, the crystallization conditions for growing better crystals in space are remarkably different from the conditions optimized on earth. This may be due to difficulties in ground optimization, which are caused by gravity-dependent phenomena, in particular the specific convective flow occurring with liquid liquid diffusion.     

Crystallization of aluminium orthophosphate

Aluminium orthophosphate crystals were crystallized by a hydrothermal technique using various solvents. The influence of nutrient materials and solvents on the rate of crystallization, solubility, morphology and quality of the crystals was studied. The crystals obtained were subjected to analysis by X-ray diffraction (XRD), scanning electron microscopy (SEM) and IR spectroscopy.     

The Biological Macromolecule Crystallization Database and NASA Protein Crystal Growth Archive

The NIST/NASA/CARB Biological Macromolecule Crystallization Database (BMCD), NIST Standard Reference Database 21, contains crystal data and crystallization conditions for biological macromolecules. The database entries include data abstracted from published crystallographic reports. Each entry consists of information describing the biological macromolecule crystallized and crystal data and the crystallization conditions for each crystal form. The BMCD serves as the NASA Protein Crystal Growth Archive in that it contains protocols and results of crystallization experiments undertaken in microgravity (space). These database entries report the results, whether successful or not, from NASA-sponsored protein crystal growth experiments in microgravity and from microgravity crystallization studies sponsored by other international organizations. The BMCD was designed as a tool to assist x-ray crystallographers in the development of protocols to crystallize biological macromolecules, those that have previously been crystallized, and those that have not been crystallized.