Ai Niino

Membrane Protein Crystallization Using Laser Irradiation

We demonstrate the crystallization of a membrane protein using femtosecond laser irradiation. This method, which we call the laser irradiated growth technique (LIGHT), is useful for producing AcrB crystals in a solution of low supersaturation range. LIGHT is characterized by reduced nucleation times. This feature is important for crystallizing membrane proteins because of their labile properties when solubilized as protein-detergent micelles. Using LIGHT, high-quality crystals of a membrane transporter protein, AcrB, were obtained. The resulting crystals were found to be of sufficiently high resolution for X-ray diffraction. The results reported here indicate that LIGHT is a powerful tool for membrane protein crystallization, as well as for the growth of soluble proteins.

Improving the quality of protein crystals using stirring crystallization

Recent reports state that a high magnetic field improves the crystal quality of bovine adenosine deaminase (ADA) with an inhibitor [Kinoshita et al.: Acta Cryst. D59 (2003) 1333]. In this paper, we examine the effect of stirring solution on ADA crystallization using a vapor-diffusion technique with rotary and figure-eight motion shakers. The probability of obtaining high-quality crystals is increased with stirring in a figure-eight pattern. Furthermore, rotary stirring greatly increased the probability of obtaining high-quality crystals, however, nucleation time was also increased. The crystal structure with the inhibitor was determined at a high resolution using a crystal obtained from a stirred solution. These results indicate that stirring with simple equipment is as useful as the high magnetic field technique for protein crystallization.

Solution stirring initiates nucleation and improves the quality of adenosine deaminase crystals.

Crystals of bovine adenosine deaminase (ADA) grown over a two-week period in the presence of an inhibitor (ADA complex) were found to be of low quality for X-ray diffraction analysis. Furthermore, ADA incubated in the absence of an inhibitor (ADA native) did not form any crystals using conventional crystallization methods. A solution-stirring technique was used to obtain high-quality ADA complex and ADA native crystals. The crystals obtained using this technique were found to be of high quality and were shown to have high structural resolution for X-ray diffraction analyses. The results reported here indicate that the solution-stirring technique promotes nucleation and improves the quality of protein crystals.

Temperature-Screening System for Determining Protein Crystallization Conditions

A newly proposed temperature-screening system has a practical application in determining protein crystallization conditions. Using a batch method, we demonstrated multitemperature conditions on one plate in a multitemperature experimental unit, providing accurate temperature control with a linear gradient. This screening system, which we call temperature at once (TAON), enables us to easily construct a solubility phase diagram and to search for the optimum temperature for crystal growth using microscale samples.

Protein crystallization by combining laser irradiation and solution-stirring techniques

Bovine adenosine deaminase in the absence of an inhibitor (free-ADA) does not form crystals when using conventional crystallization methods. Using a solution-stirring technique, we recently succeeded in generating a small number of free-ADA crystals. In this paper, we demonstrate the combination of laser-irradiated growth and stirring (COLAS). This technique was found to be useful for controlling crystal nucleation and growth, which led to the production of a much larger number of high-quality free-ADA crystals.

Control of Protein Crystal Nucleation and Growth Using Stirring Solution

We have previously developed a protein crystallization technique using a stirring protein solution and revealed that (i) continuous stirring prevents excess spontaneous nucleation and accelerates the growth of protein crystals and (ii) prestirring (solution stirring in advance) promotes the crystal nucleation of hen egg-white lysozyme. In bovine adenosine deaminase (ADA) crystallization, continuous stirring improves the crystal quality but elongates the nucleation time. In this paper, in order to control both the crystal nucleation and growth of ADA using a Micro-Stirring technique, we carried out five different stirring patterns such as (i) no stirring, (ii) continuous stirring, (iii) prestirring, (iv) poststirring (stirring late in the growth period) and (v) restirring (combined pre- and poststirring). The results showed that high-quality well-shaped crystals were obtained under the continuous stirring and restirring conditions and the nucleation time under the prestirring and restirring conditions was shorter than that under the continuous stirring and poststirring conditions. Consequently, high-quality crystals were promptly obtained under the restirring condition. These results suggest that we are able to control both the nucleation and growth of protein crystals with the stirring techniques.

A Semiautomatic Protein Crystallization System with Preventing Evaporation of Drops and Surface Sensor of Solution

We developed a simple, semiautomated protein crystallization system. The system performs crystallization-condition-screening experiments using commercial solution kits and crystallization plates. It is capable of dispensing a minimum of one microliter of protein solution into a protein well and a maximum of one milliliter of a mother liquor into a reservoir with high reproducibility using two syringes of different sizes. Several new instruments effective in preventing evaporation of solutions, a surface sensor of solutions, and a tube-holder box for solution kits are introduced.

Novel and Simple Screening Methods for Protein Crystallization by Vapor Diffusion Rate Control

We investigated the effect of the vapor diffusion rate on the refinement of protein crystals. The reservoir volume, drop-to-reservoir distance, and air space were altered to change the vapor diffusion rate using a very dense insoluble liquid and specific crystallization plates. The experimental results reveal that the drop-to-reservoir distance and air space have a more significant effect on crystal growth than the reservoir volume. We propose new simple and useful methods for screening protein crystal growth conditions based on vapor diffusion rate control.

New Practical Technique for Protein Crystallization with Floating and Stirring Methods

We previously developed an effective method for growing large, high-quality protein crystals. The method, which we call the floating and stirring technique (FAST), enables us to grow crystals on an insoluble and very dense liquid without contacting the growth vessel, and to stir a protein solution mildly. Our newly designed technique, Micro-FAST, makes FAST suitable for practical use. Micro-FAST enables us to reduce the sample volume and is applied to the vapor diffusion technique. Crystals grown by Micro-FAST demonstrated excellent X-ray diffraction with high resolution.

Pre-Stirring Promotes Nucleation of Protein Crystals

We examined the effect of stirring solutions before protein crystallization, which we call pre-stirring. We previously demonstrated that stirring the protein solution continuously promotes the effective growth of protein crystals. The continuous stirring prevents excess spontaneous nucleation and accelerates the growth of protein crystals. Subsequently, we found another effect of stirring solutions, when the stirring was discontinued. The crystallization of hen egg white lysozyme by vapor diffusion revealed that the nucleation time was determined by how the protein solution was stirred. Pre-stirring reduced nucleation time, but continuous stirring made it longer than that achieved under the nonstirred (conventional) condition. These results indicate that ideal pre-stirring promotes crystal nucleation, while continuous stirring prevents it. The choice of a stirring technique will thus enable us to precisely control the nucleation of protein crystals.