DECAMER STRUCTURE OF BOVINE PANCREATIC TRYPSIN INHIBITOR (BPTI) CRYSTALLISED FROM THIOCYANATE.
C. Hamiaux1, T. Prangé1,2, M. Riès-Kautt3, A. Ducruix3, S. Lafont3,4, J.P. Astier4 and S. Veesler4.
1LURE, Bât 209d
Université Paris-Sud, 91405 ORSAY Cedex, France.
2Chimie Structurale
Biomoléculaire (URA-1430 CNRS), UFR Biomédicale, 74, rue M.
Cachin, 93012-Bobigny Cedex, France.
3LEBS, Bât 34, UMR 9920
CNRS, Campus du CNRS, 91198 Gif sur Yvette Cedex, France.
4CRMC2, CNRS, Campus de
Luminy, Case 913, 13288 Marseille Cedex 09, France.
Bovine Pancreatic Trypsin Inhibitor is a small protein of 58 amino acids, with MW=6500 Da and pI=10.5. It usually crystallises in presence of phosphate salts at basic pH and leads to three different forms which can diffract to very high resolution (PDB entry codes 4PTI, 5PTI & 6PTI of form I, II & III respectively).
BPTI also crystallises under acid conditions (pH=4.5) in sodium chloride, potassium thiocyanate and ammonium sulfate, giving different polymorphs. Solubility diagrams are different in each case. In particular, a reverse behaviour towards temperature has been described for KSCN solution where the solubility decreases with increasing temperature. The effectiveness of the anions to crystallise BPTI follow the reverse order of the Hoffmeister series (SCN\S(-; ) > Cl\S(-; ) > SO\S(2-;4) ) [1], as observed for other basic proteins [2]. Moreover, quasi-elastic light scattering and small-angle X-ray scattering experiments were performed and show that the different solutions are monodispersed, but not monomeric in the vicinity of the solubility curve. In NaCl, (NH4)2SO4 and KSCN solutions, the scatterers have the same size (Rh ~ 23Å) and a tetramer was proposed to be the particle in solution [1].
Three new crystal forms were characterised in monoclinic (KSCN), hexagonal (NaCl and (NH4)2SO4) and orthorhombic systems ((NH4)2SO4). In each case, the diffraction limit is moderate (~ 2.7 Å) and the cell parameters are huge compared to phosphate forms. Crystals from thiocyanate were investigated. They belong to the P21 space group (a=71.56Å, b=73.83Å, c=64.47Å and ß=93.91°) and diffract up to 2.6Å resolution. Ten independent molecules were located by a multi-body molecular replacement search as developed in the AMoRe program. The molecular arrangement of the subunits is a decamer resulting from a combination of two orthogonal five-fold and two-fold non-crystallographic axes. This builds a globular, micelle-like particle which minimises hydrophobic interactions with the solvent. The refinement was conducted using XPLOR with non-crystallographic-symmetry restraints up to a final residual of R=0.20 (Rfree=0.26). Ten thiocyanate molecules were included in the final model.
This is the third structure (with
Erabutoxin-b dimers [3] and Turkey Egg-Lysozyme [4]) that reports
the presence of thiocyanate ions in the vicinity of positively
charged residues (arginines or lysines). Nevertheless, the exact
role of thiocyanate on the crystallisation process remains
unclear.
1. S. Lafont, S. Veesler, J.P.
Astier and R. Boistelle, J. Cryst. Growth 173
(1997), 132-140.
2. M. Riès-Kautt and A. Ducruix,
methods in enzymology vol. 276, 45-48
3. P. Saludjian, T. Prangé,
J. Navaza, R. Menez, J.P. Guilloteau, M. Riès-Kautt and A.
Ducruix, Acta. Cryst. B48 (1992), 520-531.
4. P.L. Howell, Acta.
Cryst. D51 (1995), 654-662.