EXPRESSION, PURIFICATION AND CRYSTALLIZATION OF DEUTERATED PROTEINS FOR NEUTRON DIFFRACTION

Expression, purification and crystallization of deuterated proteins for neutron diffraction

Kyoko Suto,^a Noritake Yasuoka,^b Masaya Kitamura^cand Hiroshi Mizuno^a

^aNational Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan; ^bFaculty of Science, Himeji Institute of Technology, Hyogo, 678-1297, Japan; ^cFaculty of Engineering, Osaka City University, Osaka 558-8585, Japan (ksuto@affrc.go.jp)

In cases of most proteins, it is not easy to obtain crystals larger than 2mm³ necessary for neutron diffraction measurement. Instead of preparation of such large crystals, we tried to prepare and crystallize deuterated proteins. Replacement of hydrogen atoms by deuterium atoms reduces the background noise and increases the scattering power in neutron diffraction. In this case, such large crystals may not be required. We’ll show expression, deuteration and crystallization FMN-binding protein (FMN-bp) using E. Coli system.

FMN-bp from Desulfovibrio vulgaris Miyazaki F is composed of 122 amino acids and a FMN, which is the smallest among known flavoproteins[1]. The function of FMN-bp in vivo is unclear at present, however it might take part in the electron-transfer pathway. Structural studies have been already carried out by X-ray crystallography[2].

Crystals of FMN-bp were grown to 2mm x 0.5mm x 0.7mm and diffracted up to 0.84 Å resolution at 100K, and to 1.10 Å at room temperature using X-ray. In the density maps, assignments of the atom species have been easily carried out, judging from the peak height in electron density map. The O, N, C atoms were identified even at side chain of aspartic acid and / or threonine. Some peaks in the difference Fourier map could be assigned for hydrogen atoms when they were involved in the hydrogen bonding, but plurality of hydrogen atoms bonding to oxygen atoms could not be clearly assigned. It was difficult to find hydrogen atoms particularly bonding to electronegative atoms or high temperature factor atoms[3]. Neutron crystallography may provide to determine above hydrogen atoms positions.

Deuteration of FMN-bp was carried out using E. Coli-OD2 D medium made by Silantes. Hi level expression system suitable to the medium was prepared using pET-20b(+) vector with E. Coli strain BL21(DE3). The deuterated FMN-bp expressed without any tags and purified by ion-exchange column and gel filtration. Purified 5mg holo FMN-bp and 3mg apo FMN-bp were obtained from 1L medium. The molecular weights of deuterated FMN-bp and non-labeled FMN-bp were measured by TOFF-MASS spectrum and deuterium ratio was estimated to 73%. Firstly crystallization of the deuterated FMN-bp was tried with conditions similar to the case of non-labeled FMN-bp. Although some crystals appeared, good crystals could not be obtained. When suitable crystallization condition was dramatically changed, the unit cell of the crystal changed from non-labeled FMN-bp. The crystals obtained in D₂O solution grew up to 1mm x 0.5mm x 0.3mm. The X-ray diffraction data was measured up to 1.35Å resolution. Preparation of larger size of crystals is now in progress for neutron diffraction study.

References

1 Kitamura, M., et al. (1994) J. Biol. Chem. 269, 5566-5573.

2 Suto, K., et al. (2000) Acta Crystallogr., D56, 368-371

3 Suto, K., et al. (2001) J. Phys. Soc. Jpn., 70, Supplement A, 406-407