Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water.

Abstract

It has become clear since about a decade ago, that the biosphere contains a variety of microorganisms that can live and grow in extreme environments. Hyperthermophilic microorganisms, present among Archaea and Bacteria, proliferate at temperatures of around 80-100 degrees C. The majority of the genera known to date are of marine origin, however, some of them have been found in continental hot springs and solfataric fields. Metabolic processes and specific biological functions of these organisms are mediated by enzymes and proteins that function optimally under these extreme conditions. We are now only starting to understand the structural, thermodynamic and kinetic basis for function and stability under conditions of high temperature, salt and extremes of pH. Insights gained from the study of such macromolecules help to extend our understanding of protein biochemistry and -biophysics and are becoming increasingly important for the investigation of fundamental problems in structure biology such as protein stability and protein folding. Extreme conditions in the biosphere require either the adaptation of the amino acid sequence of a protein by mutations, the optimization of weak interactions within the protein and at the protein-solvent boundary, the influence of extrinsic factors such as metabolites, cofactors, compatible solutes. Furthermore folding catalysts, known as chaperones, that assist the folding of proteins may be involved or increased protein protein synthesis in order to compensate for destruction by extreme conditions. The comparison of structure and stability of homologous proteins from mesophiles and hyperthermophiles has revealed important determinants of thermal stability of proteins. Rather than being the consequence of one dominant type of interactions or of a general stabilization strategy, it appears that the adaptation to high temperatures reflects a number of subtle interactions, often characteristic for each protein species, that minimize the surface energy and the hydration of apolar surface groups while burying hydrophobic residues and maximizing packing of the core as well as the energy due to charge-charge interactions and hydrogen bonds. In this article, mechanisms of intrinsic stabilization of proteins are reviewed. These mechanisms are found on different levels of structural organization. Among the extrinsic stabilization factors, emphasis is put on archaea chaperonins and their still strongly debated function. It will be shown, that optimization of weak protein-protein and protein-solvent interactions plays a key role in gaining thermostability. The difficulties in correlating suitable optimization criteria with real thermodynamic stability measures are due to experimental difficulties in measuring stabilization energies in large proteins or protein oligomers and will be discussed. Thus small single domain proteins or isolated domains of larger proteins may serve as model systems for large or multidomain proteins which due to the complexity of their thermal unfolding transitions cannot be analyzed by equilibrium thermodynamics. The analysis of the energetics of the thermal unfolding of a small, hyperthermostable DNA binding protein from Sulfolobus has revealed that a high melting temperature is not synonymous with a larger maximum thermodynamic stability. Finally, it is now well documented, that many thermophilic and hyperthermophilic proteins show a statistically increased number of salt bridges and salt bridge networks. However their contribution to thermodynamic and functional stability is still obscure.