caa a22 4500 463191331 CHVBK 20180406164910.0 cr unu---uuuuu 170326e20070901xx s 000 0 eng 10.1007/s10973-006-7961-z doi (NATIONALLICENCE)springer-10.1007/s10973-006-7961-z Membrane proteins in thin films [Elektronische Daten] A DSC study [E. Grell, A. Geoffroy, M. Stolz, E. Lewitzki, M. von Raumer] Molecular functions and structural changes of membrane proteins in an aqueous environment can be elucidated by reaction-induced FTIR difference spectroscopy upon photolysis of caged compounds. The achieved detection of IR band changes even due to single amino acid residues is, however, only possible in the presence of very high protein concentrations, implying that a low water content must be present. In general, the films are formed by controlled dehydration of membrane protein suspensions at reduced pressure and low temperature. For the retention of enzymatic activity of Na,K-ATPase, for example, a cosolvent such as glycerol is required. In order to interprete the results obtained by FTIR spectroscopy, it is important to know whether essential properties of the proteins such as hydration are changed upon film formation. Therefore, a differential scanning calorimetry (DSC) study has been carried out with purified Na,K-ATPase and Ca-ATPase in suspension, in form of pellets obtained by high-speed ultracentrifugation and in thin films. As relevant thermoanalytical properties, the endothermic denaturation transitions of the proteins have been studied. For Na,K-ATPase in the presence of 20% glycerol as cosolvent, a single, comparatively narrow endothermic and irreversible denaturation transition with a denaturation enthalpy of about 1.7 MJ mol−1 and transition temperatures of about 65 and 70°C is found in concentrated suspension and in the state of the pellet, respectively. In the case of thin films suitable for IR spectroscopy, a characteristic change is observed in a reproducible manner. The enthalpy change of the remaining transition around 70°C is reduced but an additional transition at about 77°C is observed. Based on control experiments, the new high temperature transition is attributed to a partially dehydrated state of the protein. Furthermore, a comparatively broad endothermic transition around 20°C is found under conditions of high protein concentrations (film), which is tentatively assigned to a transition of the lipid environment of this integral membrane protein. Similar results are found for Ca-ATPase films. In the absence of glycerol, the deoxycholate treated enzyme in suspension exhibits a narrow endothermic main transition at 52°C with a denaturation enthalpy around 0.9 MJ mol−1. For the film of this protein, two almost equally large endothermic transitions are found at 59 and 77°C. Also here, the data are characteristic of partial protein dehydration. These results show clearly that DSC can easily be applied in a sensitive manner to control and characterize the integrity and hydration properties of concentrated protein samples in thin films. Springer Science+Business Media LLC, 2007 Ca-ATPase nationallicence DSC nationallicence IR spectroscopy nationallicence membrane proteins nationallicence Na,K-ATPase nationallicence protein film nationallicence protein hydration nationallicence Grell E. Max-Planck-Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt, Germany aut Geoffroy A. Solvias AG, Klybeckstr. 191, 4002, Basel, Switzerland aut Stolz M. Max-Planck-Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt, Germany aut Lewitzki E. Max-Planck-Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt, Germany aut von Raumer M. Solvias AG, Klybeckstr. 191, 4002, Basel, Switzerland aut Journal of Thermal Analysis and Calorimetry Springer US; http://www.springer-ny.com 89/3(2007-09-01), 723-727 1388-6150 89:3<723 2007 89 10973 https://doi.org/10.1007/s10973-006-7961-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10973-006-7961-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Grell E. Max-Planck-Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt, Germany aut NATIONALLICENCE 700 1- Geoffroy A. Solvias AG, Klybeckstr. 191, 4002, Basel, Switzerland aut NATIONALLICENCE 700 1- Stolz M. Max-Planck-Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt, Germany aut NATIONALLICENCE 700 1- Lewitzki E. Max-Planck-Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt, Germany aut NATIONALLICENCE 700 1- von Raumer M. Solvias AG, Klybeckstr. 191, 4002, Basel, Switzerland aut NATIONALLICENCE 773 0- Journal of Thermal Analysis and Calorimetry Springer US; http://www.springer-ny.com 89/3(2007-09-01), 723-727 1388-6150 89:3<723 2007 89 10973 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer