Protein S, a calcium-binding spore coat protein from the soil bacterium Myxococcus xanthus, belongs to a group of structurally related proteins, the βγ-crystallin superfamily. Common features of this protein family are the Greek-key structural motif or crystallin fold, and the fact that all members are extremely stable long term. To investigate the correlation between the stability and Greek-key topology, protein S was cloned, expressed in Escherichia coli and purified to homogeneity. Ca 2ϩ binding influences the native tertiary structure of protein S, whereas the secondary structure remains unaffected as shown by spectroscopic methods. Ca 2ϩ ions enhance the conformational stability of protein S significantly. The midpoints of urea and guanidinium chloride-induced transitions show a difference of 1.4 M and 0.5 M denaturant, respectively, in the absence and in the presence of calcium. An equilibrium intermediate indicating independent domain folding can be detected at pH 2. In addition, thermal denaturation shows a clear deviation from the two-state model of folding, again with a strong stabilisation by Ca 2ϩ ions. Temperature and denaturant-induced equilibrium transitions are fully reversible. Our data implicate a different strategy for achieving the high stability required for the biological function compared with the structurally related lens crystallins.Keywords : Greek-key topology; βγ-crystallins; long-term stability ; protein stability; ligand binding.The βγ-crystallin superfamily comprises a ubiquitous class of proteins that are closely related in sequence as well as gene and protein structure. It is named after the β-and γ-crystallins, highly soluble structural proteins from the vertebrate eye lens [1]. They are similar two-domain proteins with an all-β structure. Each domain is formed from two Greek-key motifs that are organised as two four-stranded antiparallel β-sheets [2Ϫ4]. The conserved sequence signature led to the identification of four non-lens members of this family: the development-specific proteins spherulin 3a of the slime mould Physarum polycephalum, a single domain protein [5], and protein S from Myxococcus xanthus [6] which are both induced by stresses that trigger sporulation or encystment. The NMR-solution structures of both proteins confirmed the affiliation to this family [7Ϫ9]. An epidermal differentiation-specific protein (EDSP or ep37) of the amphibian Cynops pyrrhogaster [10,11] and AIM1, which is associated with the tumorigenicity in human malignant melanoma [12], are vertebrate representatives, the structures of which remain to be solved. The NMR structure of a killer toxin from the yeast Williopsis mrakii (WmKT) also revealed a similarity to the crystallin folding pattern, although no sequence similarity could be found [13]. WmKT may represent an interesting single-domain example of evolutionary convergence [14]. A common feature of all these proteins, besides the folding pattern, is that they belong to the most long-lived globular proteins known todayCorrespondence to E.-M. M...