High Pressure NMR Spectroscopy and its Application to the Cold Shock Protein TmCsp Derived from the Hyperthermophilic Bacterium Thermotoga maritima

Abstract

High pressure is a powerful tool to study the physico-chemical properties of proteins, especially folding, as well as the dynamics and structure of folding intermediates. Here we combine pressure with multidimensional NMR spectroscopy to characterise the physico-chemical properties of the cold shock protein TmCsp at variable temperatures. This protein is the most thermostable cold shock protein currently known and exhibits an extreme pressure stability. For this purpose we adapted a high pressure capillary system to the high field NMR spectrometers operating at 500 and 600 MHz. Since the signal to noise ratio is a limiting factor for many experiments, we devised a sapphire on-line pressure system which doubles the sample volume compared to previous systems. To understand the effect of pressure on proteins, we need to know the pressure dependence of 1H chemical shifts in random coil model tetrapeptides. The results allow to distinguish structural changes from the pressure dependence of the chemical shifts. In addition, the influence of pressure on the buffer system was investigated. Due to the limited sample volume of the high pressure capillaries it is difficult to identify NOE contacts for structure determination. Alternatively, data of residual dipolar couplings can be used for the solution of a structure under high pressure. The pressure stability of the phospholipid bicelles necessary for measurements of residual dipolar couplings is shown. 1H-15N-HSQC- and 2D- 13 C-HNCO-spectra were acquired to study the effects of pressure and temperature on chemical shifts and signal volumes of the cold shock protein TmCsp. These measurements show the high pressure stability of TmCsp compared to other proteins. In addition we studied the cold denaturation of TmCsp under 200 MPa down to a temperature of 255 K since the freezing point of water is decreased by pressure. By varying the temperature from 255 K to 340 K we observed a significant correlation between the temperature of maximum stability and the secondary structure of the protein backbone.