Rui-Qing Chen

Sensitivity of lysozyme crystallization to minute variations in concentration

It is well known that the crystallization of proteins is strongly dependent on the crystallization conditions, which are sometimes very sensitive to environmental disturbances. Parameters such as the concentration of precipitants or protein, pH, temperature and many others are known to affect the probability of crystallization, and the task of crystallizing a new protein often involves a trial-and-error test using numerous combinations of crystallization conditions. These crystallization parameters, such as the concentration of either the protein or the precipitant, are important because they directly affect the driving force of crystallization: the supersaturation of the solution. Although it is common sense that the concentration can affect the crystallization process, the sensitivity of the crystallization process to variations in the concentration has seldom been addressed. Owing to the difficulty of directly preparing solutions with very small concentration variations, it is hard to carry out an investigation of their effect on the crystallization process. In this paper, a simple but novel method for studying the effect of minute concentration variations on the success rate of protein crystallization is presented. By evaporating the crystallization droplet, a fine concentration gradient could be created. With this fine-tuned concentration gradient, it was possible to observe the effects of minute variations in the concentration or supersaturation on the crystallization. A very minor change in concentration (as low as 0.13% of the initial concentration, i.e. 0.026 mg ml(-1) for lysozyme and 0.052 mg ml(-1) for NaCl in the current study) or a very minor change in supersaturation (as small as 0.018) could cause a clear difference in the crystallization success rate, indicating that the crystallization of proteins is very sensitive to the concentration level. Such sensitive behaviour may be one reason for the poor reproducibility of protein crystallization.

An ignored variable: solution preparation temperature in protein crystallization

Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.

Replacing a reservoir solution with desiccant in vapor diffusion protein crystallization screening

This paper presents a modification to the conventional vapor diffusion (hanging- or sitting-drop) technique for protein crystallization screening. In this modified method, the reservoir solution is replaced with a desiccant to allow for a larger range of protein solution concentrations, thereby providing more opportunities for crystal formation. This method was tested in both reproducibility and screening studies, and the results showed that it significantly improves the efficiency and reduces the cost of protein crystallization screens.

Promoting protein crystallization using a plate with simple geometry

Increasing the probability of obtaining protein crystals in crystallization screening is always an important goal for protein crystallography. In this paper, a new method called the cross-diffusion microbatch (CDM) method is presented, which aims to efficiently promote protein crystallization and increase the chance of obtaining protein crystals. In this method, a very simple crystallization plate was designed in which all crystallization droplets are in one sealed space, so that a variety of volatile components from one droplet can diffuse into any other droplet via vapour diffusion. Crystallization screening and reproducibility tests indicate that this method could be a potentially powerful technique in practical protein crystallization screening. It can help to obtain crystals with higher probability and at a lower cost, while using a simple and easy procedure.

A gradual desiccation method for improving the efficiency of protein crystallization screening

In vapor diffusion protein crystallization screening, it has been reported that replacing the reservoir solution with desiccant can increase the crystallization success rate. Therefore, the desiccation method is a potentially powerful method in practical protein crystallization screening. However, this method is difficult to apply broadly because the optimal amount of desiccant for a specific screening task is unknown. Utilizing an unsuitable amount of desiccant can result in even worse screening results than would be obtained from the traditional vapor diffusion method. Here, it is shown that by employing a modified strategy, named the gradual desiccation method, the problem can be solved without knowing the optimal amount of desiccant, and the crystallization success rate can be further increased compared with the one-time desiccation method.

An investigation of the effects of varying pH on protein crystallization screening

Protein crystallization occurs when the solution conditions are suitable for nucleation and growth of the crystals. pH, as one of the most important parameters that governs the protein crystallization process, can affect the conformation, activity, electrostatic interactions and solubility of protein in the solution. Hence, manipulating the pH is an important path to success in crystallizing proteins. In this paper, we show that varying pH levels over the incubation time during the crystallization process can help increase the chance of obtaining protein crystals. The process of varying the pH can be considered as one way to automatically search for a suitable pH for successful crystallization. This discovery can guide us to develop a new screening strategy in which varying the pH against the incubation time is the major process used during the crystallization screening. Our screening experiments verified that varying the pH is indeed a promising method to achieve efficient crystallization screening of a protein.

Sensitivity of lysozyme crystallization to temperature variation

Environments with varying temperatures have been shown to beneficially increase the probability of obtaining protein crystals. Therefore, a cycling temperature strategy (CTS) has been proposed for protein crystallization screening. During the practical application of this strategy, it is necessary to know the effective temperature range that promotes crystallization to design a suitable temperature program. In this paper, the effects of different temperature ranges on lysozyme crystallization (or more specifically, nucleation) were investigated. The results show that a small periodic variation in the temperature range of as little as 0.4 K can have a significant effect on the crystallization success rate under some crystallization concentration conditions, confirming that crystallization of lysozyme is very sensitive to temperature variation. Because practical protein crystallization is always performed in an environment with slight temperature variations, the sensitivity of protein crystallization to temperature may provide an explanation for the poor reproducibility of protein crystallization. Further investigation of the CTS on lysozyme crystallization showed that a cycling temperature strategy exerts an effect on protein crystallization by altering the supersaturation caused by changes in temperature.

Measurement of contact angles in a simulated microgravity environment generated by a large gradient magnetic field

The contact angle is an important parameter that is essential for studying interfacial phenomena. The contact angle can be measured using commercially available instruments. However, these well-developed instruments may not function or may be unsuitable for use in some special environments. A simulated microgravity generated by a large gradient magnetic field is such an environment in which the current measurement instruments cannot be installed. To measure the contact angle in this environment, new tools must be designed and manufactured to be compatible with the size and physical environment. In this study, we report the development and construction of a new setup that was specifically designed for use in a strong magnetic field to measure the contact angle between a levitated droplet and a solid surface. The application of the setup in a large gradient magnetic field was tested, and the contact angles were readily measured.

Effect of the weather conditions during solution preparation on lysozyme crystallization

Protein crystallization is a delicate process that is always sensitive to environmental factors. When the environmental factors are not well controlled or not controlled at all, identical crystallization droplets from the same mother liquid may yield different crystallization results. One environmental factor, the weather conditions during crystallization solution preparation, is not usually considered as a parameter for protein crystallization. In this paper, it is shown that the weather parameters during preparation of the crystallization experiment, including the ambient temperature, humidity, pressure and particulate matter in the air, can all affect the reproducibility of lysozyme crystallization. An identical lysozyme crystallization experiment was repeated for an entire year, and the weather conditions when each crystallization experiment was set up were recorded along with the crystallization results. Among the parameters recorded, the humidity during the experiment setup showed the strongest effect on lysozyme crystallization. On the basis of these results, it is suggested that the weather conditions during crystallization solution preparation should be considered as a potential factor that can influence protein crystallization.

Research Progresses on Structure and Functions of the Cytoplasmic Polyadenylation Element Binding Proteins

The cytoplasmic polyadenylation element binding proteins(CPEB) are mRNA-binding proteins,and play great roles in invertebrates and vertebrates.In this paper,the research progresses on structure and biological functions of CPEB were reviewed,and the research results of the CPEB in translation repression and activation of mRNA,oogenesis,cellular senescence,development,and synaptic plasticity in neuron were summarized.The molecular mechanisms of related biological incidents were discussed.In the end,the research prospects of CPEB were presented.