The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system.

Each protein folds into a unique and native structure spontaneously. However, during the unfolding or refolding process, a protein often tends to form aggregates. To establish a method to prevent undesirable protein aggregation and to increase the stability of native protein structures under deterioration conditions, two types of aggregation conditions, thermal unfolding-induced aggregation and dilution-induced aggregation from denatured state, were studied in the presence of additional amino acids and ions using lysozyme as a model protein. Among 15 amino acids tested, arginine exhibited the best results in preventing the formation of aggregates in both cases. Further biophysical studies revealed that arginine did not change the thermal denaturation temperature (T(m)) of the lysozyme. The preventive effect of arginine on aggregation was not dependent on the size or isoelectric point of eight kinds of proteins tested.

The crystal structure of aspartyl-tRNA synthetase (AspRS) from Pyrococcus kodakaraensis was solved at 1.9 Å resolution. The sequence and three-dimensional structure of the catalytic domain are highly homologous to those of eukaryotic AspRSs. In contrast, the N-terminal domain, whose function is to bind the tRNA anticodon, is more similar to that of eubacterial enzymes. Its structure explains the unique property of archaeal AspRSs of accommodating both tRNA Asp and tRNA Asn . Soaking the apo-enzyme crystals with ATP and aspartic acid both separately and together allows the adenylate formation to be followed. Due to the asymmetry of the dimeric enzyme in the crystalline state, different steps of the reaction could be visualized within the same crystal. Four different states of the aspartic acid activation reaction could thus be characterized, revealing the functional correlation of the observed conformational changes. The binding of the amino acid substrate induces movement of two invariant loops which secure the position of the peptidyl moiety for adenylate formation. An unambiguous spatial and functional assignment of three magnesium ion cofactors can be made. This study shows the important role of residues present in both archaeal and eukaryotic AspRSs, but absent from the eubacterial enzymes.

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Proteins tend to form inactive aggregates at high temperatures. We show that polyamines, which have a relatively simple structure as oligoamids, effectively prevent thermal inactivation and aggregation of hen egg lysozyme. In the presence of additives, including arginine and guanidine (100 mM), more than 30% of 0.2 mgAEmL )1 lysozyme in sodium phosphate buffer (pH 6.5) formed insoluble aggregates by heat treatment (98°C for 30 min). 1However, in the presence of 50 mM spermine or spermidine, no aggregates were observed after the same heat treatment. The residual activity of lysozyme after this heat treatment was very low (< 5%), even in the presence of 100 mM arginine and guanidine, while it was maintained at 50% in the presence of 100 mM spermine and spermidine. These results imply that polyamines are new candidates as molecular additives for preventing the thermal aggregation and inactivation of heat-labile proteins.

The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1␣ plays a central role in its targeting to chromatin. Recombinant HP1␣ prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1␣ was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1␣'s affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1␣ to H3K9me by mediating the interaction between HP1␣ and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1␣ exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1␣'s N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.The formation of the higher-order chromatin structure, the so-called heterochromatin, is critical for genomic stability and transcriptional silencing (19,21,30). Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein that was initially discovered in Drosophila melanogaster as a factor that is enriched at highly condensed, transcriptionally inert regions of polytene chromosomes (24,25). HP1 is an evolutionarily conserved molecule whose homologues and isoforms have been previously identified in a wide range of eukaryotic organisms (9). As well as being an architectural component of heterochromatin, HP1 is known to play crucial roles in heterochromatin-mediated gene silencing. Profound effects of HP1 deletion, mutation, or overexpression on transcriptional silencing have been previously demonstrated in Drosophila (11, 12), Schizosaccharomyces pombe (13), and mammals (17). Moreover, an ever-expanding list of HP1-interacting proteins indicates that HP1 may also be involved in the regulation of several biological processes, including transcriptional activation, faithful chromosome segregation, and DNA damage repair (8,19,21,30).Structurally, all HP1 homologues possess two functionally distinct globular domains, the N-terminal chromodomain (CD) (39) and the C-terminal chromoshadow domain (CSD) (1), which are linked by an unstructured hinge region. The CD recognizes and interacts with methylated lysine 9 of the histone H3 N-terminal tail, which is a characteristic modification in heterochromatic regions (4,27,35). The CSD, on the other hand, is responsible for dimer formation and is involved in HP1's interactions with a large variety of proteins (5,44,46). Because of its abilities to bind directly to chromatin an...

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