THE EAFP CRYSTAL GROWTH OBSERVED BY ATOMIC FORCE MICROSCOPY

Sheng Wang,^a,b Ye Xiang,^b Genpei Li^b and Dacheng Wang^b

^aBioengineering Institute of Chongqing University, Chongqing, 400044, P.R. China; ^bInstitute of Biophysics and National Microgravity Lab., CAS, 100101, P.R. China (wtscrystal@sina.com)

Eucommia antifungal protein(EAFP) is one kind of antifungal protein[1]. EAFP has 41 residues and crystals of EAFP belong to space group P2₁ with unit-cell parameters a=19.085, b=23.225, c=30.854, b=98.64 [2]. EAFP crystals grew in only 4-5 hours much faster than most of other macromolecular crystals. It is noteworthy that the EAFP crystals diffract to almost the limiting resolution (~0.8 Å). The 3D structure was determined by direct methods with SnB[3,4] and refined with Shelx-97 [5].

The EAFP crystals were grown according to the method of Mr. Xiang[2]. The target crystals grown directly on a glass cover glass were moved onto the AFM sample stage for the acquisition of AFM images. All the AFM images were collected on the {100} faces of the EAFP crystals in a fluid cell filled with mother liquor by both the contact and non-contact modes.

Nucleation and dynamic growth were studied by in situ Atomic Force Microscopy (Autoprobe CP-Research, Park Scientific Instruments). We found that EAFP had special aggregates, ring-shaped clusters in original solution. The diameter varied from about 180nm to 350nm. During AFM experiments we also found that many linear aggregates sedimented on the surface of EAFP and then incorporated into the lattice of the crystals. So the ring-shaped aggregates might be split by precipitator and absorbed on the surface directly or be connected with each other into longer linear aggregates.

At higher supersaturation we found that the EAFP crystals grew very fast with macro steps by two-dimensional nucleation. The growing steps developed by groups which consisted of about 5-6 steps. The step height was about 1.9 nm corresponding well with the length of one axis. At low-to-moderate supersaturation, one type of apparently anisotropic single-double-like spiral dislocations was found on the {100} faces of EAFP crystals. After 8-9 screw steps all of them would automatically grow into a series of whole intact elliptic layers. Thus we concluded that during the process of growth EAFP molecules had strong ability to adjust themselves to fit the lattice which perhaps was related to the special character of EAFP molecules.

This work intended to understand the nucleation of EAFP crystal growth and kinetic mechanisms. This will help and maybe guide us to choose crystal conditions for obtaining good quality crystals to give higher diffraction ability, and shed light on our knowledge concerning protein crystallization.

References

1 Huang, R.-H., Xiang, Y., Liu, X.-Zh., et al. (2002) FEBS Letters, 521: 87-90.

2 Xiang, Y., Huang, R.-H., Liu W., et al. (2002) Acta Cryst.D58, 1838-1840.

3 Weeks, C.M. & Miller, R. (1999) J. Appl. Cryst. 32, 120-124.

4 Blessing, R.H., & Smith, G.D. (1999) J. Appl. Cryst. 32, 664-670.

5 Sheldrick, G.M.(1997)SHELX97, University of Göttingen, Germany.