Stabilisation of protein structures by solvent molecules

Ľ. Urbániková and J. Ševčík 

Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovak Republic, e-mail: lubica.urbanikova@savba.sk; jozef.sevcik@savba.sk

RNase Sa, Sa2 and Sa3, ribonucleases produced by various strains of Streptomyces aureofaciens, have proven to be excellent models for various types of studies including structure-function relationship, cytotoxicity, mechanism of enzymatic reaction, protein-protein and protein-nucleic acid recognition. They are small proteins consisting of 96-98 amino-acid residues with 56-69 % identity in primary structures. Properties of the enzymes have been thoroughly studied. More than ten years of structural studies of S.a. ribonucleases resulted in solving a number of 3D structures of free enzymes [1,2,3], mutants and complexes with several ligands at various resolutions including atomic. Structures of ten RNase Sa mutants have been used for the study of conformational stability of globular proteins orientated on better understanding of the contribution of individual hydrogen bonds to the protein stability [4,5,6]. Mutations were prepared with the aim to remove a limited number of hydrogen bonds at retaining the protein structure. It has been proved that the effect of a single amino-acid mutation on conformational stability of a protein highly depends on the location of the substitution and its environment in the structure.

High accuracy structures refined against high resolution data (2.0 - 1.0 Å) allowed inspection of subtle changes not only in protein itself but also in the solvent structure. There are evidences that protein-water hydrogen bonds contribute to the stability of the protein molecule. In the structures of all three S.a. ribonucleases and RNase Sa mutants, water molecules which are fully or mostly buried in the protein structures were identified. Their possible role in the stabilisation of protein structure will be discussed.

RNase Sa, Sa2, Sa3 and RNase Sa mutants were crystallised usually from phosphate buffer using ammonium sulphate as precipitant or additive, therefore it is not surprising that sulphate/phosphate anions were identified also in the structures. The structures of sulphate and phosphate anions are nearly identical; therefore the identity of the anions cannot be clarified. Their possible role in mediating crystal contacts and stabilization of protein molecules will be discussed. Structural observations will be extrapolated on behaviour of the enzymes in solution to support experimental findings on the stabilising role of phosphate buffer used in biochemical measurements and isolation of S.a. ribonucleases.

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