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.

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.

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.

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.

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%.

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

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.

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.

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.

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.

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.

Protein Characteristics and Field Efficacy of YN1-1, a Highly Virulent Strain of Bacillus thuringiensis

[Objective] This study aimed to investigate the biological characteristics of a Bacillus thuringiensis strain YNI-1, which has high virulence to Lepidoptera spp. [Method] The crystal protein of YNI-1 was analyzed by SDS-PAGE, and its indoor and field efficacy for Lepidoptera spp. was investigated. [Result] The parasporal crystal of YNI-1 has a diamond-like structure. The molecular weight of the original toxin protein is 136 kDa. After trypsin treatment, the original toxin protein was hy- drolyzed into active toxin protein with molecular weight of 63 kDa. For Plutella xy- Iostella and Pieris rapae, the indoor efficacy of B. thuringiensis was better than that of commercial B. thuringiensis （WP）. In view of field efficacy, rate of YNI-1 strain was higher than that of commercial B [Conelusion] YNI-1 strain has excellent development potential. the insects reduced thuringiensis （WP）.

Toxicity of Bacillus thuringiensis Crystal Toxins to Field Populations of Rice Leaf Folder, Cnaphalocrocis medinalis （Guenee） and Establishment of Baseline Susceptibility to Cry1Ab

Eight insecticidal crystal proteins of Bacillus thuringiensis, CrylAa, CrylAb, CrylAc, CrylB, Cry2Aa, CrylC, CrylDa and Cry 1Ea were assessed for toxicity against 1 st instar larvae of rice leaf folder, Cnaphalocrocis medinalis （Guenee） at 48 HAT and 72 HAT. Bioassay results depicted CrylAa was the most toxic （LCso 2.35 ppm） followed by CrylBa （LCso 8,50 ppm） and CrylAb （LCso 8.73 ppm） at 48 HAT, whereas, at 72 HAT CrylAb proved to be highly toxic （LC50 0.50 ppm） followed by CrylAa （LCso 4.07 ppm）, CrylAc （LCso 4,84 ppm） and CrylBa （LCso 6.42 ppm）. Toxins Cry2Aa, CrylCa, CrylDa and CrylEa did not resulted in any mortality at 48 HAT and 72 HAT, respectively. Baseline estimates for CrylAb against 1st instar larvae of C. medinalis sampled from seven geographical locations revealed variation in LC50＇s from 0.37 ppm to LC50 16.25 ppm at 48 HAT and LC50 0.50 ppm to LC50 6.49 ppm 72 HAT, respectively with relative resistance ratios of 44-fold and 13-fold at 48 HAT and 72 HAT over the susceptible population.

Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

Myelin basic protein（MBP） is an essential structure involved in the generation of central nervous system（CNS）myelin.Myelin shape has been described as liquid crystal structure of biological membrane.The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin.In this paper,we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy（AFM）.By analyzing the pressure–area（π–A） and pressure–time（π–T） isotherms,univariate linear regression equation was obtained.In addition,the elastic modulus,surface pressure increase,maximal insertion pressure,and synergy factor of monolayers were detected.These parameters can be used to modulate the monolayers binding of protein,and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3-phosphoserine（DPPS） monolayer,followed by DPPC/DPPS mixed and1,2-dipalmitoyl-sn-glycero-3-phospho-choline（DPPC） monolayers via electrostatic and hydrophobic interactions.AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP（5 n M） show a phase separation texture at the surface pressure of 20 m N/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure.MBP is not an integral membrane protein but,due to its positive charge,interacts with the lipid head groups and stabilizes the membranes.The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value.

Structural analysis of tumor-relatedsingle amino acid mutations in human MxA protein

Background:Human myxovirus resistant protein A(MxA),encoded by the myxovirus resistance 1(Mx1) gene,is an interferon(IFN)-triggered dynamin-like multi-domain GTPase involved in innate immune responses against viral infections.Recent studies suggest that MxA is associated with several human cancers and may be a tumor suppressor and a promising biomarker for IFN therapy.Mxl gene mutations in the coding region for MxA have been discovered in many types of cancer,suggesting potential biological associations between mutations in MxA protein and corresponding cancers.In this study,we performed a systematic analysis based on the crystal structures of MxA and elucidated how these mutations specifically affect the structure and therefore the function of MxA protein.Methods:Cancer-associated Mxl mutations were collected and screened from the COSMIC database.Twenty-two unique mutations that cause single amino acid alterations in the MxA protein were chosen for the analysis.Amino acid sequence alignment was performed using Clustal W to check the conservation level of mutation sites in Mx proteins and dynamins.Structural analysis of the mutants was carried out with Coot.Structural models of selected mutants were generated by the SWISS-MODEL server for comparison with the corresponding non-mutated structures.All structural figures were generated using PyMOL.Results:We analyzed the conservation level of the single-point mutation sites and mapped them on different domains of MxA.Through individual structural analysis,we found that some mutations severely affect the stability and function of MxA either by disrupting the intraVinter-molecular interactions supported by the original residues or by incurring unfavorable configuration alterations,whereas other mutations lead to gentle or no interference to the protein stability and function because of positions or polarity features.The potential clinical value of the mutations that lead to drastic influence on MxA protein is also assessed.Conclusions:Among all of the reported tumor-associated single-point mutations,seven of them notably affect the structure and function of MxA and therefore deserve more attention with respect to potential clinical applications.Our research provides an example for systematic analysis and consequence evaluation of single-point mutations on a given cancer-related protein.

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.

Study of Growth Mechanism of Lysozyme Crystal by Batch Crystallization Method

The lysozyme crystals were made by batch crystallization method and the distribution of aggregate in solution were measured by dynamic light scattering. The results showed that the dimension of aggregate increased with the increase of the concentration of lysozyme and NaCl, lysozyme molecules aggregated gradually in solution and finally arrived at balance each other. The higher the concentrations of lysozyme and NaCl were, the faster the growth rate of （110） face was. The growth rates of lysozyme crystal were obtained by a Zeiss microscope, and the effective surface energy （a） of growing steps were calculated about 4.01×10^-8s J·cm^-2 according to the model of multiple two-dimensional nucleation mechanism.