Ren-Bin Zhou

A strategy for selecting the pH of protein solutions to enhance crystallization

The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.

A review on recent advances for nucleants and nucleation in protein crystallization

The elucidation of protein structures by X-ray crystallography remains the most effectual method to provide accurate structural details at atomic resolution for rational drug design and other biotechnological research studies. Also, emerging applications of protein crystals as ordered nanostructure scaffolds for catalysis, imaging, and drug delivery are attracting much attention. However, the first step of these applications is obtaining high-quality crystals, which is still an obstacle. Successful crystallization requires two steps: nucleation and crystal growth, while the nucleation is a precondition for harvesting the crystal of interest. So controlling protein nucleation may be an alternative breakthrough for this bottleneck. It is well known that nucleants can induce protein crystallization and improve crystal quality, so investigation on the nucleants that can be universally used for any protein crystallization is ongoing. This manuscript reviews the advances that have been achieved using nucleants in protein crystallization and it is a suitable reference for practical crystallization.

A protein crystallisation screening kit designed using polyethylene glycol as major precipitant

Crystallisation of proteins is usually achieved with the help of chemical agents. Because there are few general guidelines in determining what agents will help to crystallise a specific protein, suitable crystallisation agents are often found via exhaustive trial-and-error tests by mixing many chemical agents (the collection of which is called a crystallisation screening kit) one-by-one with the protein. Currently, many commercially available crystallisation screening kits have been developed and utilised in practical crystallisation screen experiments. However, information regarding the design of new screening kits has yet to be expanded using a large amount of experimental data. Here, we show the step-by-step design processes of a polyethylene glycol-based screening kit. It was found that the screening performance could be improved by modifying the crystallisation screening kits according to the accumulated data (such as those in the Biological Macromolecule Crystallisation Database (BMCD)), the screening test results and existing knowledge. The screening kit designed in this paper can be used for practical protein crystallisation screen experiments and the method can be used in the design of other crystallisation screening kits.

Myocyte enhancer factor 2C and its directly-interacting proteins: A review

Myocyte enhancer factor 2C (MEF2C) is a transcription factor of MADS box family involved in the early development of several human cells including muscle (i.e., skeletal, cardiac, and smooth), neural, chondroid, immune, and endothelial cells. Dysfunction of MEF2C leads to embryo hypoplasia, disorganized myofibers and perinatal lethality. The main role of MEF2C is its regulation of muscle development. It has been reported that MEF2C-knockout mice die on embryonic day 9.5 from unnatural development of cardiovascular. The effects of MEF2C are mediated by its directly-interacting proteins; therefore, the investigation of these interactions is critical in order to clarify MEF2Cs biological function. In this study, we review twenty-five proteins that directly interact with MEF2C, including nineteen proteins related to muscle development, four proteins related to neural cell development, one protein related to chondroid cell development, four proteins related to immune cell development, and two proteins related to endothelial cell development. Among these proteins, the interaction of MEF2C with MRFs is important for differentiation of developing muscle cells. MEF2C interacts with Sox18 for endothelial vessel morphogenesis. The interaction of MEF2C with Cabin1 is important for maintaining T-cell inactivation. Investigating the interactions of MEF2C and its directly-interacting proteins is not only helpful to understand of the physiological function of MEF2C, but also provides a target for future rational drug design.

RNA binding motif protein 3: a potential biomarker in cancer and therapeutic target in neuroprotection

RNA binding motif 3 (RBM3) is a highly conserved cold-induced RNA binding protein that is transcriptionally up-regulated in response to harsh stresses. Featured as RNA binding protein, RBM3 is involved in mRNA biogenesis as well as stimulating protein synthesis, promoting proliferation and exerting anti-apoptotic functions. Nowadays, accumulating immunohistochemically studies have suggested RBM3 function as a proto-oncogene that is associated with tumor progression and metastasis in various cancers. Moreover, emerging evidences have also indicated that RBM3 is equally effective in neuroprotection. In the present review, we provide an overview of current knowledge concerning the role of RBM3 in various cancers and neuroprotection. Additionally, its potential roles as a promising diagnostic marker for cancer and a possible therapeutic target for neuro-related diseases are discussed.

Correlation between Protein Sequence Similarity and Crystallization Reagents in the Biological Macromolecule Crystallization Database

The protein structural entries grew far slower than the sequence entries. This is partly due to the bottleneck in obtaining diffraction quality protein crystals for structural determination using X-ray crystallography. The first step to achieve protein crystallization is to find out suitable chemical reagents. However, it is not an easy task. Exhausting trial and error tests of numerous combinations of different reagents mixed with the protein solution are usually necessary to screen out the pursuing crystallization conditions. Therefore, any attempts to help find suitable reagents for protein crystallization are helpful. In this paper, an analysis of the relationship between the protein sequence similarity and the crystallization reagents according to the information from the existing databases is presented. We extracted information of reagents and sequences from the Biological Macromolecule Crystallization Database (BMCD) and the Protein Data Bank (PDB) database, classified the proteins into different clusters according to the sequence similarity, and statistically analyzed the relationship between the sequence similarity and the crystallization reagents. The results showed that there is a pronounced positive correlation between them. Therefore, according to the correlation, prediction of feasible chemical reagents that are suitable to be used in crystallization screens for a specific protein is possible.

A Systematic Analysis of the Structures of Heterologously Expressed Proteins and Those from Their Native Hosts in the RCSB PDB Archive.

Recombinant expression of proteins has become an indispensable tool in modern day research. The large yields of recombinantly expressed proteins accelerate the structural and functional characterization of proteins. Nevertheless, there are literature reported that the recombinant proteins show some differences in structure and function as compared with the native ones. Now there have been more than 100,000 structures (from both recombinant and native sources) publicly available in the Protein Data Bank (PDB) archive, which makes it possible to investigate if there exist any proteins in the RCSB PDB archive that have identical sequence but have some difference in structures. In this paper, we present the results of a systematic comparative study of the 3D structures of identical naturally purified versus recombinantly expressed proteins. The structural data and sequence information of the proteins were mined from the RCSB PDB archive. The combinatorial extension (CE), FATCAT-flexible and TM-Align methods were employed to align the protein structures. The root-mean-square distance (RMSD), TM-score, P-value, Z-score, secondary structural elements and hydrogen bonds were used to assess the structure similarity. A thorough analysis of the PDB archive generated five-hundred-seventeen pairs of native and recombinant proteins that have identical sequence. There were no pairs of proteins that had the same sequence and significantly different structural fold, which support the hypothesis that expression in a heterologous host usually could fold correctly into their native forms.

Application of protein crystallization methodologies to enhance the solubility, stability and monodispersity of proteins

Protein crystallization screens are commonly used to find specific crystallization conditions. In practice, undesirable clear drops are often ignored. We hypothesize that clear drops may be potentially useful for finding the conditions leading to proteins with high solubility and monodispersity . Based on this idea, we first tested three model proteins and confirmed that improved monodispersity could be observed in the clear drops. Next, we used this strategy to achieve the maximum solubility and monodispersity of a new protein (RNA binding motif 3, RBM3). We suggest that this strategy is useful not only for high-throughput screening of optimal solubilization conditions but also for screening of biopharmaceutical formulations.

Effects of large gradient high magnetic field (LG-HMF) on the long-term culture of aquatic organisms: Planarians example: Planarian Regeneration in Simulated Gravity

Large gradient high magnetic field (LG-HMF) is a powerful tool to study the effects of altered gravity on organisms. In our study, a platform for the long-term culture of aquatic organisms was designed based on a special superconducting magnet with an LG-HMF, which can provide three apparent gravity levels (µ g, 1 g, and 2 g), along with a control condition on the ground. Planarians, Dugesia japonica, were head-amputated and cultured for 5 days in a platform for head reconstruction. After planarian head regeneration, all samples were taken out from the superconducting magnet for a behavioral test under geomagnetic field and normal gravity conditions. To analyze differences among the four groups, four aspects of the planarians were considered, including head regeneration rate, phototaxis response, locomotor velocity, and righting behavior. Data showed that there was no significant difference in the planarian head regeneration rate under simulated altered gravity. According to statistical analysis of the behavioral test, all of the groups had normal functioning of the phototaxis response, while the planarians that underwent head reconstruction under the microgravity environment had significantly slower locomotor velocity and spent more time in righting behavior. Furthermore, histological staining and immunohistochemistry results helped us reveal that the locomotor system of planarians was affected by the simulated microgravity environment. We further demonstrated that the circular muscle of the planarians was weakened (hematoxylin and eosin staining), and the epithelial cilia of the planarians were reduced (anti-acetylated tubulin staining) under the simulated microgravity environment. Bioelectromagnetics. 2018;39:428-440.

Ligand-binding characterization of simulated β-adrenergic-like octopamine receptor in Schistocerca gregaria via progressive structure simulation

It is important to design insecticides having both low drug resistance and less undesirable toxicity for desert locust control. Specific GPCRs of Schistocerca gregaria, especially β-adrenergic-like octopamine receptor (SgOctβR), can be considered as its potential effective insecticide targets. However, either the unavailability of SgOctβRs structure or the inadequate capability of its sequence lead the development of insecticide for Schistocerca gregaria meets its plateau. To relax this difficulty, this paper develops a promising progressive structure simulation from SgOctβRs sequence, to its predicted structure of SgOctβR in vacuum, to its conformation as well as its complex with endogenous ligand octopamine in a solvent-membrane system. The combined approach of multiple sequence alignment, static structural characterization, and dynamic process of conformational change during binding octopamine reveal three important aspects. The first one is the characterization of SgOctβRs active pocket, including the attending secondary structure elements, its hydrophobic residues and nonpolar surface. The second one is the interaction with octopamine, especially the involved hydrogen bonds and an aromatic stacking of pi-pi interactions. The third one is the potential binding sites, including six highly conserved residues and one highly variable residue for locust insecticide design. This work is definitely helpful for the further structure-based drug design for efficient and eco-friendly insecticides, as well as site-directed mutagenesis biochemical research of SgOctβR.