Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography

•A unique crystallographic studies of wild-type and mutated form of the same protein under high pressure.•Compressibility of RNase A molecule is significantly affected by a single amino acid substitution.•High pressure protein crystallography helps understanding protein flexibility and identify conformational substrates.

Hydrostatic pressure in range 0.1-1.5 GPa is used to modify biological system behaviour mostly in biophysical studies of proteins in solution. Due to specific influence on the system equilibrium high pressure can act as a filter that enables to identify and investigate higher energy protein conformers. The idea of the presented experiments is to examine the behaviour of RNase A molecule under high pressure before and after introduction of destabilizing mutation. For the first time crystal structures of wild-type bovine pancreatic ribonuclease A and its markedly less stable variant modified at position Ile106 were determined at different pressures. X-ray diffraction experiments at high pressure showed that the secondary structure of RNase A is well preserved even beyond 0.67 GPa at room temperature. Detailed structural analysis of ribonuclease A conformation observed under high pressure revealed that pressure influences hydrogen bonds pattern, cavity size and packing of molecule.

Graphical abstractDownload high-res image (98KB)Download full-size image