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MINIREVIEW SERIES Protein hyperthermostability – current status and beyond Sotirios Koutsopoulos Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA The discovery of hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 C, has revolutionized our ideas about the upper limit of temperature at which life can exist. The characterization of hyperthermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function retained at high temperatures where ‘normal’ proteins undergo dramatic structural changes? The general consensus has been that protein hyperthermostability does not involve any aberrant features but rather is accomplished through modifying only the distribution of structural features (i.e. extended ion pair networks, increased packing density, decreased number of surface loops, prevalence of specific amino acids in the sequence, etc.) that stabilize proteins which are adjusted to other environmental conditions. This series contains four articles encompassing different approaches to, and aspects of, protein hyperthermostability. In the first article, Matsui and Harata analyze crystallographic data from homologous mesophilic, thermophilic and hyperthermophilic proteins and discuss the importance of buried polar interactions. It has been long suggested that ion pair interactions are essential to stabilize the protein structure at high temperatures. Herein, it is proposed that ion pairs in the core are more important than those on the surface of hyperthermostable proteins. In the second review, Luke and colleagues carefully distinguish between hyperthermophilic and thermophilic proteins and compare them with their mesophilic counterparts. Thermodynamic and kinetic data of protein unfolding in vitro reveal remarkable differences: the study concludes that hyperthermostability is primarily linked to very slow protein unfolding kinetics. This implies that hyperthermophiles survived by selection of protein mutants that unfold slowly. The third review by Tehei and Zaccai addresses the role of dynamics on protein stability. Protein atoms are not fixed, as depicted in crystal structures, but fluctuate. Hence, the whole protein fluctuates as well. The dynamic nature of hyperthermostable proteins may be the key to unraveling the mechanism responsible for the delicate balance between rigidity, which is related to heat resistance, and molecular fluctuations at high temperatures, which account for biological function. In the last article, Unsworth and colleagues review current theories and suggest that a combination of structural, dynamic and other physicochemical attributes are optimized to ensure stability and activity at high temperatures. The potential for utilizing heat stable proteins was demonstrated in the PCR reaction, a revolutionary technique in molecular biology. This review also summarizes methodologies and proposes strategies for improving heat stability and activity of hyperthermostable proteins for applications in biocatalysis and biotechnology. The reviews presented here highlight the significant advances made to date towards understanding protein stability and function at high temperatures, but also raise questions. New discoveries have pushed the limits of hyperthermostability to higher temperatures; is it now necessary to consider as hyperthermostable only those proteins that are stable at temperatures near 100 C and above? In aqueous media above 100 C, hydrophobic interactions and hydrogen bonds are significantly weakened; is this the reason why such interactions are not frequently observed as stabilizing factors in hyperthermostable proteins? To date, data have been collected from in vitro studies of dilute protein solutions; are the conclusions valid for intracellular proteins where biomolecular crowding is an important stabilizing factor? Structural information for hyperthermostable proteins is derived from diffraction of crystals grown at (or below) room temperature; do these crystal structures represent the structure of these proteins in their native high temperature environment? Advances in protein science will continue to generate more systematic structural and physicochemical information on hyperthermostable proteins and the features that underlie their unique properties. Sotirios Koutsopoulos is a Senior Research Fellow in the Center for Biomedical Engineering at the Massachusetts Institute of Technology (MIT). He received his undergraduate degree in chemistry from the University of Patras, Greece and completed his PhD in the same institute in physical chemistry of biological phenomena. He conducted postdoctoral work in the Danish Technical University in nanotechnology and then, as a Marie Curie fellow, in Wageningen University, the Netherlands, studied biophysics, specifically analyzing factors involved in the thermal stability and biological function of hyperthermostable proteins. Dr Koutsopoulos joined MIT in 2005 to study protein stability and misfolding in cells. doi: 10.1111/j.1742-4658.2007.05957.x FEBS Journal 274 (2007) 4011 ª 2007 The Author Journal compilation ª 2007 FEBS 4011