Cold-shock proteins (Csps) are a subgroup of the coldinduced proteins preferentially expressed in bacteria and other organisms on reduction of the growth temperature below the physiological temperature. They are related to the cold-shock domain found in eukaryotes and are some of the most conserved proteins known. Their exact function is still not known, but translational regulation, possibly via RNA chaperoning, has been discussed.Here we present the structure of a hyperthermophilic member of the Csp family. The NMR solution structure of TmCsp from Thermotoga maritima, the hyperthermophilic member of this class of proteins, was solved on the basis of 1015 conformational constraints. It contains five b strands combined in two antiparallel b sheets making up a b barrel structure, in which b strands 1±4 are arranged in a Greekkey topology. The side chain of R2, which is exclusively found in thermophilic members of the Csp family, probably participates in a peripheral ion cluster involving residues D20, R2, E47 and K63, suggesting that the thermostability of TmCsp is based on the peripheral ion cluster around the side chain of R2.Keywords: cold-shock protein; hyperthermophiles; NMR spectroscopy; structure determination; Thermotoga maritima.The cold-shock response in micro-organisms is a transient phenomenon affecting the growth rate of the cell and the saturation of fatty acids as well as the rate of synthesis of DNA, RNA, and protein at temperatures significantly lower than the normal physiological temperature [1]. For most proteins, the expression under cold shock is dramatically decreased, whereas for a few it is increased. Of the latter, one group of small acidic proteins shows an extremely high induction level and high affinity to single stranded nucleic acids [2]. Because of their high sequence homology, they are classified together in the family of cold-shock proteins (Csps) (reviewed in [3±5]). The biological function is still not known, but translational regulation, possibly via RNA chaperoning, is the possibility currently favored [6].The first hyperthermophilic member of this family has been cloned from Thermotoga maritima (TmCsp); its sequence and physicochemical characteristics were recently reported [7]. TmCsp is a small globular protein of 66 amino acids with a molecular mass of 7474 Da. It exhibits extreme intrinsic stability making it the most thermostable Csp known at present [7].The Csps with known 3D structure are Greek-key b barrel proteins and belong to the`OB-fold family' . For EcCspA, the binding specificity has not yet been determined; for ssDNA the sequences CCAAT and ATTGG seem to be preferred while ssRNA is bound unspecifically. A thorough analysis of the interaction of BsCspB with ssDNA templates revealed that BsCspB preferentially binds to polypyrimidine but not polypurine ssDNA templates [2]. Thymine-based ssDNA templates bind with high-affinity and salt-independently, whereas binding of cytosine-based ssDNA templates is strongly salt-dependent, indicating that a large electrostatic c...