On the stability of proteins from hyperthermophiles : a structural and thermodynamic study

Abstract: In this study the structural and thermodynamic basis of the thermal stability of proteins has been studied using proteins from hyperthermophilic organisms as model systems. A new type of chaperonin (TF55) has been isolated from the hyperthermophilic archaeon Sulfolobus solfataricus consisting of two different subunits named TF55-alpha and TF55-beta. Two dimensional projections of electron microscopy images revealed a nine fold symmetrical complex. Two rings of nine subunits are stacked upon each other forming a cage-like complex of about 17.5 nm in height and 16 nm in diameter with a central cavity of 4.5 nm. Significant structural changes have been observed after phosphorylation of the chaperonin which has been found to be modulated by potassium and magnesium ions. A small basic protein (Sso7d) has been isolated from the hypethermophilic archaeon Sulfolobus solfataricus and its three dimensional structure has been determined by NMR-spectroscopy. The solution structure of Sso7d comprises a triple stranded beta-sheet onto which an orthogonal double stranded beta-sheet is packed. Sso7d binds strongly to double stranded DNA and protects it from thermal denaturation. A model of a Sso7d DNA complex has been proposed on the basis of NMR measurements and the electrostatic properties of the protein The thermal unfolding of Sso7d has been studied by CD-spectroscopy and differential scanning calorimetry. Maximum stability has been observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 97.7 and 98.7 °C and an unfolding enthalpy of about 64 kcal/mol. The analysis of the thermodynamic data measured revealed that, despite its high melting temperature, Sso7d is not a protein with a high intrinsic stability. Its high melting temperature results from a flattening of its stability curve indicating that thermal stability is achieved on the cost of the thermodynamic stability of the protein. The crystal structure of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been determined at 3 Å resolution and has been compared to the structures of glutamate dehydrogenases from the mesophilic bacterium Clostridium symbiosum and the hyperthermophilic archaeon Pyrococcus furiosus. The comparison revealed that common as well as distinct mechanisms contribute to the thermal stability of the two thermostable enzymes. Common mechanisms observed in the two thermostable enzymes are 1.) the increase of the number of ion pairs; 2.) the increase in the extent of ion pair networks; 3.) the reduction of the number and volume of cavities in the six subunits. Striking differences have been observed at the subunit interfaces of both thermostable enzymes. In P. furiosus glutamate dehydrogenase the subunit contacts are dominated by ionic interactions realized by large salt bridge networks. In T. maritima however, the number of intersubunit salt bridges is reduced and subunits contacts are stabilized by hydrophobic rather than electrostatic interactions.