The fluid phenomena in the crystallization of the protein crystal

This paper reports that an optical diagnostic system consisting of Mach-Zehnder interferometer with a phase shift device and image processor has been used for study of the kinetics of protein crystal growing process. The crystallization process of protein crystal by vapour diffusion is investigated. The interference fringes are observed in real time. The present experiment demonstrates that the diffusion and the sedimentation influence the crystallization of protein crystal which grows in solution, and the concentration capillary convection associated with surface tension occurs at the vicinity of free surface of the protein mother liquor, and directly affects on the outcome of protein crystallization. So far the detailed analysis and the important role of the fluid phenomena in protein crystallization have been discussed a little in both space- and ground-based crystal growth experiments. It is also found that these fluid phenomena affect the outcome of protein crystallization, regular growth, and crystal quality. This may explain the fact that many results of space-based investigation do not show overall improvement.

Direct observation of bunching of elementary steps on protein crystals under forced flow conditions

Bunching of elementary steps by solution flow is still not yet clarified for protein crystals. Hence, in this study, we observed elementary steps on crystal surfaces of model protein hen egg-white lysozyme （HEWL） under forced flow conditions, by our advanced optical microscopy. We found that in the case of a HEWL solution of 99.99% purity, forced flow changed bunched steps into elementary ones （debunching） on tetragonal HEWL crystals. In contrast, in the case of a HEWL solution of 98.5% purity, forced flow significantly induced bunching of elementary steps. These results indicate that in the case of HEWL crystals, the mass transfer of impurities is more significantly enhanced by forced solution flow than that of solute HEWL molecules. We also showed that forced flow induced the incorporation of microcrystals into a mother crystal and the subsequent formation of screw dislocations and spiral growth hillocks.

Imaging protein crystal growth behaviour in batch cooling crystallisation

The temporal and spatial growth behaviour of protein crystals, subject to different cooling strategies in protein crystallisation was investigated. Although the impact of temperature and cooling rate on crystal growth of small molecules was well documented, much less has been reported on their impact on the crystallisation of proteins. In this paper, an experimental set-up is configured to carry out such a study which involves an automatic temperature controlled hot-stage crystalliser fitted with a real-time imaging system. Linbro parallel crystallisation experiments(24-well plate) were also conducted to find the suitable initial conditions to be used in the hot-stage crystallisation experiments, including the initial concentration of HEW lysozyme solutions, precipitate concentration and pH value. It was observed that fast cooling rates at the early stage led to precipitates while slow cooling rates produced crystal nuclei, and very slow cooling rates, much smaller than for small molecules are critical to the growth of the nuclei and the crystals to a desired shape. The interesting results provide valuable insight as well as experimental proof of the feasibility and effectiveness of cooling as a means for achieving controlled protein crystallisation, compared with the evaporation approach which was widely used to grow single large crystals for X-ray diffraction study. Since cooling rate control can be easily achieved and has good repeatability, it suggests that large-scale production of protein crystals can be effectively achieved by manipulating cooling rates.

Sequence-Based Protein Crystallization Propensity Prediction for Structural Genomics: Review and Comparative Analysis

Structural genomics (SG) is an international effort that aims at solving three-dimensional shapes of important biological macro-molecules with primary focus on proteins. One of the main bottlenecks in SG is the ability to produce dif-fraction quality crystals for X-ray crystallogra-phy based protein structure determination. SG pipelines allow for certain flexibility in target selection which motivates development of in- silico methods for sequence-based prediction/ assessment of the protein crystallization pro-pensity. We overview existing SG databanks that are used to derive these predictive models and we discuss analytical results concerning protein sequence properties that were discov-ered to correlate with the ability to form crystals. We also contrast and empirically compare mo- dern sequence-based predictors of crystalliza-tion propensity including OB-Score, ParCrys, XtalPred and CRYSTALP2. Our analysis shows that these methods provide useful and compli-mentary predictions. Although their average ac- curacy is similar at around 70%, we show that application of a simple majority-vote based en-semble improves accuracy to almost 74%. The best improvements are achieved by combining XtalPred with CRYSTALP2 while OB-Score and ParCrys methods overlap to a larger extend, although they still complement the other two predictors. We also demonstrate that 90% of the protein chains can be correctly predicted by at least one of these methods, which suggests that more accurate ensembles could be built in the future. We believe that current protein crystalli-zation propensity predictors could provide useful input for the target selection procedures utilized by the SG centers.

Joint Operation of Atomic Force Microscope and Advanced Laser Confocal Microscope for Observing Surface Processes in a Protein Crystal

We demonstrated the insitu observation of a moving atomic force microscope (AFM) cantilever using a laser confocal microscope combined with a differential interference microscope (LCM-DIM). The AFM cantilever scanned or indented the {110} surface of a hen egg-white lysozyme crystal in a supersaturated solution. Using a soft cantilever, we could observe the step growth with high time resolution by LCM-DIM and perform quantitative measurements of the step height by AFM simultaneously. In addition, a hard cantilever was used with LCM-DIM to observe the dynamics of crystal surface scratching and indentation. In the supersaturated solution, the small steps generated from the scratched line aggregated to macro steps, and subsequently flattened the surface.

Positive and negative effects of graphite flake and monolayer graphene oxide templates on protein crystallization

Heterogeneous template-induced nucleation is a promising way to regulate protein crystallization events and could be employed for purification processes and crystallographic studies.Protein crystallization process with graphite and graphene oxide,as heterogeneous templates,were investigated.More than 640 hanging drops with different concentrations of Lysozyme(30,50,70,100 mg/mL)and NaCl(0.7,0.9,1.1,1.3,1.5 M)were crystallised at 4 ℃ with or without graphite/graphene oxide templates.The induction times and crystallization process were observed under the microscope.The lysozyme in the solutions with graphite flakes nucleated faster under all the conditions than the lysozyme with equal experimental conditions without templates.The crystals preferred to grow around the edge of graphite flakes than on the flat surfaces.In the droplets with monolayer graphene oxide,more crystals appeared around gra-phene oxide particles,and the faster or slower nucleation processes with templates were dependent on the lysozyme and NaCl concentrations.Graphene oxide templates strongly inhibited nucleation at high lysozyme concentrations but promoted nucleation at low lysozyme concentrations.Both heterogeneous templates changed the crystal morphology and the crystallization kinetics.More crystals were observed in the solution with graphite templatesthan with graphene oxide templates and without any template.

Influence of microgravity on protein crystal structures

Structural determination and comparison of microgravity and ground grown protein crystals have been carried out in order to investigate the effect of microgravity on the structure of protein crystals. Following the structural studies on the hen egg-white lysozyme cystals grown in space and on the ground, the same kind of comparative studies was performed with acidic phospholipase A2 crystals grown in different gravities. Based on the results obtained so far, a conclusion could be made that microgravity might not be strong enough to change the conformation of polypeptide chain of proteins, but it may improve the bound waters’ structure, and this might be an important factor for microgravity to improve the protein crystal quality. In addition, the difference in the improvement between the two kinds of protein crystals may imply that the degree of improvement of a protein crystal in microgravity may be related to the solvent content in the protein crystal.

The second space experiment of protein crystallization with domestic facilities

The second experiment of protein crystallization was performed on domestic re-entry satelliteFSW-2 in 1994-07. The results are superior to the ones of the first mission in 1992: 9 of 10 different proteins were crystallized in space, and 70% of the total 48 samples yielded single crystals. Besides hen egg-white lysozyme which grew high-quality crystals on the first mission, an acidic phospholipase A2(aPLA2) from snake venom and hemoglobin from Anser Indicus produced good-quality crystals suitable for X-ray diffraction analyses. The positive effect of microgravity on protein crystal growth is verified again at this time.

Protein Crystal Growth in Microgravity

Protein crystal growth is quite important for the determination of protein structureswhich are essential to the understanding of life at molecular level as well as to the development of molecu-lar biotechnology.The microgravity environment of space is an ideal place to study the complicated pro-tein crystallization and to grow good-quality protein crystals.A number of crystal-growth experiments of10 different proteins were carried out in August,1992 on the Chinese re-entry satellite FSW-2 in spaceusing a tube crystallization equipment made in China.A total of 25 samples from 6 proteins producedcrystals,and the effects of microgravity on protein crystal growth were observed,especially for an acidicphospholipase A2 and henegg-white lysozyme which gave better crystals in space than earth-grown crys-tals in ground control experiments.The results have shown that the microgravity in space favors the im-provement of the size,perfection,morphology and internal order of the grown protein crytals.

Cellulose-based Polymeric Liquid Crystals as a Biomimetic Modifier for Suppressing Protein Adsorption

A novel biomimetic protein-resistant modifier based on cellulose-based polymeric liquid crystals was described（PLCs）. Two types of PLCs of propyl hydroxypropyl cellulose ester（PPC） and octyl hydroxypropyl cellulose ester（OPC） were prepared by esterification from hydroxypropyl cellulose, and then were mixed with polyvinyl chloride and polyurethane to obtain composite films by solution casting, respectively. The surface morphology of PLCs and their composite films were characterized by polarized optical microscopy（POM） and scanning electron microscopy（SEM）, suggesting the existence of microdomain separation with fingerprint texture in PLC composite films. Water contact angle measurement results indicated that hydrophilicity of PLC/polymer composite films was dependent on the type and content of PLC as well as the type of matrix due to their interaction. Using bovine serum albumin（BSA） as a model protein, protein adsorption results revealed that PLCs with protein-resistant property can obviously suppress protein adsorption on their composite films, probably due to their flexible LC state. Moreover, all PLCs and their composites exhibited non-toxicity by MTT assay, suggesting their safety for biomedical applications.

Observation on the modifying activity of the protein from Elytrzgia repens rhizome for ice crystal

In winter, spring and summer, the rhizome of wild Elytrzgia repens of Heilongjiang Province was selected to extract the soluble which whole protein and the apoplastic protein, and analyzed by SDS-PAGE. The result indicated that there were two specific polypeptides in two types protein from winter; their relative molecular weight were identified as 52 ku and 26 ku by analyzing software; the apoplastic protein from winter had the ability of modifing the growth of ice crystal which appeared hexagonal in shape observed with the phase-contrast photomicroscope. So the apoplastic protein from winter has the antifreeze characters and the 52 ku protein is more likely the antifreeze protein

Cloning, Purification, Crystallization and Preliminary X-Ray Diffraction Studies of Periplasmic Glucose Binding Protein of <i>Pseudomonas putida</i>CSV86

Biochemical data and genomic analysis indicate the involvement of a putative ABC transporter for glucose transport in Pseudomonas putida CSV86. The periplasmic solute binding proteins are known to confer substrate specificity to the ABC transporters by binding specifically to the substrate and transferring them to their cognate inner membrane transport assembly. Periplasmic glucose binding protein from Pseudomonas putida CSV86 (ppGBP) was found to be glucose specific. The gene encoding ppGBP was cloned. Recombinant ppGBP was overexpressed and purified to homogeneity. The purified recombinant protein showed glucose binding activity of 752 pmol/mg of protein and was crystallized as a complex with glucose. The crystal diffracted to 1.7 &Aring resolution using home X-ray source. Preliminary analysis of diffraction data showed that the crystals belonged to space group P21212 with unit-cell parameters a = 102.56, b = 119.2, c = 66.65 &Aring and α = β = γ = 90&deg. Matthews coefficient calculation showed the presence of two molecules in the asymmetric unit with solvent content of 45.7%.

RECENT DOMESTIC PROGRESS IN SPACE CRYSTALLIZATION OF PROTEINS

The advances of protein crystal growth in microgravity are limited by its low success rate of space crystallization experiments. Our recent efforts have concentrated on exploration of the ways to increase the success rate of the experiments.The corresponding studies include structural comparisons of space- and Earthgrown protein crystals, numerical simulations of solute transport in protein crystallizer, optimization of protein crystailization conditions and improvement of crystallization techniques used. These studies show that the success rate of space protein crystallization could be improved by different ways.

A review of factors affecting the success of membrane protein crystallization using bicelles

Several reports have been published detailing various platforms for obtaining crystals of membrane proteins to determine their structure including those that use disk shaped bilayers called bicelles. While these crystals have been readily grown and used for X-ray diffraction, the general understanding as to why bicelles are adequate for such a procedure or how to rationally choose conditions remains unknown. This review intends to discuss issues of protein stabilization and precipitation in the presence of lipids that may influence crystal formation.

Crystallization and Preliminary Crystallographic Studies of Luffaculin 1,a Ribosome-inactivating Protein from the Seeds of Luffa Acutangula

Luffaculin 1 purified from the seeds of Luffa acutangula belongs to the type I ribosome-inactivating proteins （RIPs）. It has been crystallized by the vapor-diffusion method using polyethylene glycol as a precipitant. The crystal is of space group P1 with a = 39.135, b = 46.813, c = 83.571 A, α = 891068,β = 80.009 and y = 72.143°, and has two molecules per asymmetric unit. X-ray data have been collected to be 1.4 A using a synchrotron source.

Prediction of Crystallization Propensity of Proteins from <i>Bacillus haloduran</i>Using Various Amino Acid and Protein Features

Correct prediction of propensity of crystallization of proteins is important for cost- and time-saving in determination of 3-demensional structures because one can focus to crystallize the proteins whose propensity is high through predictions instead of choosing proteins randomly. However, so far this job has yet to accomplish although huge efforts have been made over years, because it is still extremely hard to find an intrinsic feature in a protein to directly relate to the propensity of crystallization of the given protein. Despite of this difficulty, efforts are never stopped in testing of known features in amino acids and proteins versus the propensity of crystallization of proteins from various sources. In this study, the comparison of the features, which were developed by us, with the features from well-known resource for the prediction of propensity of crystallization of proteins from Bacillus haloduran was conducted. In particular, the propensity of crystallization of proteins is considered as a yes-no event, so 185 crystallized proteins and 270 uncrystallized proteins from B. haloduran were classified as yes-no events. Each of 540 amino-acid features including the features developed by us was coupled with these yes-no events using logistic regression and neural network. The results once again demonstrated that the predictions using the features developed by us are relatively better than the predictions using any of 540 amino-acid features.

Correlating Combined Features of Amino Acid and Protein with Crystallization Propensity of Proteins from <i>Mycobacterium tuberculosis</i>

Since a decade ago, both protein and amino acid features have been correlated with crystallization propensity of proteins in order to develop methods to predict whether a protein can be crystallized. In this continuing study, each of three features combining features of amino acid and protein, was correlated with the crystallization propensity of proteins from Mycobacterium tuberculosis using logistic and neural network models. The results showed that two combined features, amino acid distribution probability and future composition, had good predictions on whether a protein would be crystallized in comparison with the predictions obtained from each of 531 amino acid features. The results obtained from the third combined feature, amino acid pair predictability, demonstrated the trend of crystallization propensity in proteins from Mycobacterium tuberculosis.

Correlation of Combined Characters of Amino Acid and Whole Protein with Success Rate of Crystallization of <i>Lactobacillus</i>Proteins

Crystallization of proteins is a very delicate process, which is influenced by many known and unknown factors. Of tested factors, many factors are exclusively related to individual amino-acid characters such as molecular weight or protein characters such as protein length. It is considered necessary to test factors that combine both individual amino-acid characters and protein characters with respect to success rate of crystallization. In this study, two combined characters characterizing individual amino-acid character and protein character, amino acid distribution probability and future composition, were used to correlate the success rate of crystallization of proteins from Lactobacillus via modeling. The results obtained from logistic regression and neural network were compared against the results obtained from each of 533 individual amino-acid characters. This study demonstrated that the combined characters are involved in crystallization process and should be taken into account for predicting the success rate of crystallization process.

Crystallization and Preliminary Crystallographic Studies of Cucurmosin 2,a Ribosome-inactivating Protein from the Sarcocarp of Cucurbita moschata

Cucurmosin 2, a type 1 ribosome-inactivating protein （RIP） isolated from sarcocarp of Cucurbita moschata, has been crystallized by the vapor-diffusion method using PEG6000 as the precipitant. The crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 55.853, b = 65.507, c = 91.754 А, and have one molecule per asymmetric unit. X-ray data have been collected to 1.8А, using a synchrotron source.

Effects of Small Biomolecules on Lysozyme Crystallization

To develop appropriate biocompatible nucleants,glutathione(GSH),glycine(Gly)and cysteine(Cys)were used as the biomolecular additives to study their effect on the regulation of lysozyme crystallization.Characterized by Fourier transform infrared spectroscopy,powder X-ray diffraction,circular dichroism,etc.,the results show that GSH can effectively promote protein crystallization under even lower concentrations of lysozyme by enhancing the nucleation rate,comparing with the additives of Gly and Cys,whereas lysozyme crystal structures produced in the presence of the biomolecular additives are similar to those in the absence of the additives.It shows in combination with molecular modeling that the stronger interactions between small biomolecular additives and the lysozyme LOOP accelerate the heterogeneous nucleation.It is suggested that such small biomolecules can be used as promising nucleants for promoting protein crystallization in the food and pharmaceutical industries.