Temperature- and pressure-induced unfolding and refolding of ubiquitin: a static and kinetic Fourier transform infrared spectroscopy study.
Temperature- and pressure-induced denaturation of the protein ubiquitin was investigated using FT-IR spectroscopy. On the basis of IR spectral parameters, different states are distinguished and a pressure/temperature-stability diagram of the protein has been determined. The evolution of the secondary structures with temperature illustrates that the band intensities of disordered structures decrease at the expense of the formation of intermolecular beta-sheets at 83 degrees C, pD 7, and ambient pressure, with the population of intramolecular beta-sheets and alpha-helices remaining essentially unchanged. At ambient temperature (T = 21 degrees C) and pD 7, ubiquitin denatures at 5.4 kbar. Contrary to other proteins studied so far, features of secondary structure of ubiquitin remain distinct at high pressure, suggesting that part of this small protein rearranges and does not unfold to disordered structures. The secondary structural changes during compression and decompression are fully reversible, and no aggregation occurs. With corresponding measurements of the pressure-induced denaturation of ubiquitin at different temperatures, a p/T-stability diagram of ubiquitin could be obtained. Furthermore, kinetic FT-IR measurements were carried out using the pressure-jump relaxation technique. The denaturation process is shown to occur on a time scale which is about twice as long as that of the renaturation process, and both processes are much slower than the unfolding-refolding kinetics observed at ambient pressure.
Herberhold
Heinz
H
Physical Chemistry I, University of Dortmund, Otto Hahn Strasse 6, D-44227 Dortmund, Germany.
Winter
Roland
R
D000577:Amides; D007700:Kinetics; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry*
D000577:Amides; D025801:Ubiquitin