[Frontiers in Bioscience 5, d787-795, September 1, 2000] 787 PROTEIN STABILITY IN EXTREMOPHILIC ARCHAEA Roberto Scandurra 1 , Valerio Consalvi 1 , Roberta Chiaraluce 1 , Laura Politi 1 , Paul C. Engel 2 1 Dipartimento di Scienze Biochimiche ”A.Rossi-Fanelli” Università ’La Sapienza’, P.le A.Moro 5 00185 Rome Italy, 2 Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Protein stability 3.1. Thermodynamic aspects of protein stability 3.2. Proteins from (hyper)thermophiles 3.3. Proteins from psychrophiles 4. Structural determinants of protein stability 4.1. Primary structure analysis 4.2. Structural adaptations 5. Conclusions 6. Perspectives 7. References 1. ABSTRACT Extremophilic microorganisms have adapted their molecular machinery to grow and thrive under the most adverse enviromental conditions. These microorganisms have found their natural habitat at the boiling and freezing point of water, in high salt concentration and at extreme pH values. The extremophilic proteins, selected by Nature to withstand this evolutionary pressure, represent a wide research field for scientists from different disciplines and the study of the determinants of their stability has been an important task for basic and applied research. A surprising conclusion emerges from these studies: there are no general rules to achieve protein stabilization. Each extremophilic protein adopts various strategies and the outstanding adaptation to extreme temperature and solvent conditions is realized through the same weak electrostatic and hydrophobic interactions among the ordinary amino acid residues which are also responsible for the proper balance between protein stability and flexibility in mesophilic proteins. 2. INTRODUCTION Microorganisms that live under extreme conditions of life, e.g.close to the freezing point or the boiling point of water, are termed ‘extremophiles’. The extreme environments, probably similar, in some cases, to those that existed during early periods of life on earth, have provided this name and are an essential part of the growth conditions for these microorganisms. The extremophilic microorganisms so far known belong to different taxa within the archaeal and bacterial domains, and the majority of these ‘exotic’ microorganisms are found within the Archaea. Representative among them are the hyperthermophile Pyrococcus furiosus (optimal growth temperature above 100°C), the thermoacidophile Sulfolobus acidocaldarius (growth temperature 80°C at pH <2.75), the alkaliphile Natronobacterium pharaonis (optimal growth at pH > 10), the halophile Haloferax mediterranei (living at >10% salt) and the psychrophile Cenarchaeum symbiosum (growth only below 25°C). The phylogenetic tree constructed on the basis of the 16/18 SrRNA sequence (1), shows a primary tripartite division of the living world into the three domains of Bacteria, Archaea and Eukarya (2). The deepest branches and the shortest lineages represent the most extreme hyperthermophiles known so far (Pyrodictium, Methanopyrus, Aquifex). These Archaea live in the hottest places on earth and are considered to be the oldest living organisms. To thrive in such adverse conditions evolution has devised some peculiar mechanisms such as the formation of very resistant macromolecular structures able to defend the archaeal cell from the hostile environment. Evolutionary pressure has, for instance, selected for the Archaea particular cell membranes more resistant than those of mesophiles. In particular, the lipid bilayer, whose fatty acids are commonly bound to glycerol molecules through ester linkages in mesophiles, is replaced by a monolayer membrane highly resistant to heat, acid and alkali, with glycerol molecules bound by ether linkages to branched hydrocarbons of the phytanyl or biphytanyl type. Another difference is that the central carbon atom of the glycerol is in the R stereoisomeric form in Bacteria and Eukarya and in the L form in Archaea. Accordingly, not only the cell membrane but all the archaeal macromolecular cellular components are expected to be highly resistant to physical and chemical stresses and possibly assembled from stable chemical compounds. Proteins from Eukarya and Bacteria, extremophilic or otherwise, are composed of amino acids linked by strong covalent bonds which