Analysis of Archaeal Cold-Adapted Proteins: Comparative Studies of the Elongation Factor 2 Proteins from Psychrotrophic, Mesophilic and Thermophilic Archaea
Torsten THOMAS* and Rick CAVICCHIOLI
School of Microbiology and Immunology, The University of NSW, Sydney, NSW, 2052, Australia
Cold-adapted enzymes are thought to exhibit structural differences compared to their mesophilic or thermophilic counterparts in order to obtain sufficient flexibility for catalysis under reduced temperature. This is based on studies of bacterial and eucaryotic proteins, and to date, comparative studies have not be performed on archaeal proteins.
Methanococcoides burtoniii was isolated from Ace Lake, Antarctica where the waters are a constant 1-2℃. It has a growth temperature maximum of 28℃ and a predicted growth temperature minimum of-2.5℃ (1). Clearly M. burtonii is adapted to growth and survival at low temperature extremes.
To reveal structural and functional features of archaeal cold-adapted proteins we sequenced the elongation factor 2 (EF-2) gene (aef2) from psychrotrophic, (M. burtonii; Topt: 23℃), mesophilic (Methanococcoides methylutens; Topt: 35℃) and thermophilic (Methanosarcina thermophila; Topt: 50℃) archaea that form a close phylogenetic cluster (16S-rRNA identity >90%).
The aef2-genes contain 2190 bp (corresonding to 730 AA) and the analysis of gene-sequences down- and up-stream of the aef2-genes suggest that they are organized in a streptomycin (str)-operon similar to Escherichia coli. Analysis of the amino acid-sequences revealed that the proteins are generally similar, although specific differences in amino acid-composition are evident. An alignment of all three elongation factor proteins showed a significant number of unique amino acid-residues; 41, 37, 144 residues for M. burtonii, M. methylutens and M. thermophila, respectively. Subsequent three dimensional-modeling of various domains of the archaeal elongation factors with they crystal structure of the bacterial Thermus thermophila EF-G, revealed that most unique residues had little effect on the topology of the predicted structures. Interestingly however, a few specific residues appeared to cause structural changes that may be associated with thermal activity. These data indicate that a limited number of amino acid-changes may be sufficient to confer adaptation to the elongation factors in accordance with the environments that they were isolated from.
To further characterize the structural and functional properties of these elongation factors we are currently over-expressing and purifying the proteins (preliminary results will be presented).
1. Franzmann, P.D., Springer, N., Ludwig, W., Conway de Macario, E., and Rhode, M. (1992) System. Appl. Microbiol., 15, 573-581.
