High pressure crystal structure of ribonuclease A
|Krzysztof Lewinski 1, Katarzyna Kurpiewska 1, Kamil F. Dziubek 2, Andrzej Katrusiak 2, Josep Font 3, Marc Ribo 3, Maria Vilanova 3|
1. Jagiellonian University, Faculty of Chemistry, Ingardena 3, Kraków 30-060, Poland
Two different factors, high pressure and mutation, can act as perturbants very useful to study several biological aspects of protein, including folding/unfolding processes, stability, oligomerization and aggregation. Detailed analysis of inter and intramolecular interactions and factors that affects them is important for understanding mechanism of structural changes being the origin of molecular diseases.
To determine the influence of high pressure and mutations on protein structure for crystallographic experiments we selected RNase A, protein that is small, easy to crystallize and can form oligomers1. Trigonal crystals of wild-type ribonuclease A (WT RNase A) and its mutated variant I106A were loaded into the Diamond Anvil Cell and pressurized in the presence of crystallization solution. The pressure was monitored using a ruby chip2. Diffraction data were collected on Nonius KappaCCD diffractometer with standard Mo sealed tube (55 kV, 30 mA) for one crystal of WT RNase A at 0.5 and 0.7 GPa, and for two crystals of I106A RNase A at 0.35 and 0.48 GPa. All four high pressure (HP) structures were solved and refined with resolution between 2.4–2.8 Å. To find out structural changes being result of high pressure, the structures were compared with the ambient pressure (AP) structures determined by us using the same diffractometer and radiation.
The most important differences introduced to the structure by the high pressure were observed for the main hydrophobic cavity volume. Cavities observed in WT HP structures are visibly smaller (VHP WT I = 20.8 Å3) from that one observed in AP structure (VAP WT = 30.8 Å3). Cavity formed in HP I106A structures (VHP I106A I = 109.6 Å3, VHP I106A II = 68.6 Å3) also differ from the cavity found in AP I106A (VAP I106A = 80.9 Å3).
In all cases the overall HP structures of WT and I106A RNase A were indistinguishable from AP structures. Nevertheless, calculation of difference distance matrices has revealed subtle rearrangements of some regions of ribonuclease molecule present in all HP structures. Magnitude of rearrangements suggests that changes observed for HP WT structures are enhanced by introduced mutation. The HP structures allowed to identify five flexible regions of RNase A molecule that can move against each other. Moreover, some of those regions were previously reported as a fragments that play curial role in high pressure unfolding of RNase A in solution3,4.
This work was undertaken within the frame of COST Action D30. It was partially funded by grants T09A 108 30 from Ministry of Science and Higher Education (Poland) and WRBW/6/2007 from Jagiellonian University, Faculty of Chemistry (Poland).
1. Gotte G., Laurents D.V., Liobonati M., Biochimica et Biophysica Acta, 2006, 1764, 44
2. Piermarini G.J., Block S., Barnet J.D., Forman R.A., J. Appl. Phys., 1975, 46, 2774
3. Navon A., Ittah V., Scheraga H.A., Haas E., Biochemistry, 2002, 41, 14225
4. Zhang J., Peng X., Jonas A., Jonas J., Biochemistry, 1995, 27, 8631
Presentation: Poster at COST D30 Final Evaluation Meeting, by Krzysztof Lewinski
See On-line Journal of COST D30 Final Evaluation Meeting
Submitted: 2007-10-08 15:59 Revised: 2009-06-07 00:48
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