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.

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.

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）.

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.

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.

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.

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.

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.

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.

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.

The recombinant expression systems for structure determination of eukaryotic membrane proteins

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryoUc membrane protein structures have been obtained due to the technical challenges in the genera- tion of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.

Synthesis of crystals and particles by crystallization and polymerization in droplet-based microfluidic devices

The recent advances in crystallization and polymerization assisted by droplet-based microfluidics to synthesize micro-particles and micro-crystals are reviewed in this paper.Droplet-based microfluidic devices are powerful tools to execute some precise controls and operations on the flow inside microchannels by adjusting fluid dynamics parameters to produce monodisperse emulsions or multiple-emulsions of various materials.Major features of this technique are producing particles of monodispersity to control the shape of particles in a new level,and to generate droplets of diverse materials including aqueous solutions,gels and polymers.Numerous microfluidic devices have been employed to generate monodisperse droplets of range from nm toμm,such as T junctions,flow-focusing devices and co-flow or cross-flow capillaries.These discrete,independently controllable droplets are ideal microreactors to be manipulated in the channels to synthesize the nanocrystals,protein crystals,polymer particles and microcapsules.The generated monodisperse particles or crystals are to meet different technical demands in many fields,such as crystal engineering,encapsulation and drug delivery systems.Microfluidic devices are promising tools in the synthesis of micron polymer particles that have diverse applications such as the photonic materials,ion-exchange and chromatography columns,and field-responsive rheological fluids.Processes assisted by microfluidic devices are able to produce the polymer particles(including Janus particles)with precise control over their sizes,size distribution,morphology and compositions.The technology of microfluidics has also been employed to generate core-shell microcapsules and solid microgels with precise controlled sizes and inner structures.The chosen“smart”materials are sensitive to an external stimulus such as the change of the pH,electric field and temperature.These complex particles are also able to be functionalized by encapsulating nanoparticles of special functions and by attaching some special groups like targeting ligands.The nucleation kinetics of some crystals like KNO_(3)was investigated in different microfluidic devices.Because of the elimination of the interactions among crystallites in bulk systems,using independent droplets may help to measure the nucleation rate more accurately.In structural biology,the droplets produced in microfluidic devices provide ideal platforms for protein crystallization on the nanoliter scale.Therefore,they become one of the promising tools to screen the optimal conditions of protein crystallization.

Enhanced and tunable fluorescent quantum dots within a single crystal of protein

The design and synthesis of bio-nano hybrid materials can not only provide new materials with novel properties, but also advance our fundamental understanding of interactions between biomolecules and their abiotic counterparts. Here, we report a new approach to achieving such a goal by growing CdS quantum dots （QDs） within single crystals of lysozyme protein. This bio-nano hybrid emitted much stronger red fluorescence than its counterpart without the crystal, and such fluorescence properties could be either enhanced or suppressed by the addition of Ag（I） or Hg（II）, respectively. The three-dimensional incorporation of CdS QDs within the lysozyme crystals was revealed by scanning transmission electron microscopy with electron tomography. More importantl~ since our approach did not disrupt the crystalline nature of the lysozyme crystals, the metal and protein interactions were able to be studied by X-ray crystallography, thus providing insight into the role of Cd（II） in the CdS QDs formation.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

Purification of L-asparaginase II by crystallization

Here a case study ofL-asparaginase II out of a recombinant Escherichia coli is presented. The target protein was obtained by simple cell disintegration and acetone precipitation. The L-asparaginase II has been crystallized in three different forms in the following microbatch crystallization. The rod-shaped crystals （-400 μm edge length） were obtained at either 8℃ or 22℃ after 17h by addition of PEG6ooo. The rectangular-shaped crystals were obtained after further recrystallization of the rod-shaped crystals. The rhombic-shaped crystals formed at 8℃ after 12 days when cold ethanol was used instead of PEG6000. All crystallizations were performed in tris-acetate buffer （50mmol-L-1, pH 5.1）. By crystallization, the specific activity of L-asparaginase II has increased 5-fold. The protein content and the purity of the crystals were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. The more concentrated L- asparaginase II out of an extract mixture and the presence of only less minor proteins after crystallization demonstrates that crystallization is an effective and mild method to purify the target protein. The single crystal X-ray diffraction pattern reveals that the crystals are proteins and the X-ray powder diffraction （XRPD） pattern shows clearly that the crystals forming in PEG600o and ethanol have different crystal structures.