yII-crystallin from calf eye lens consists of two homologous domains, connected by a six-residue linker peptide. In order to study the intrinsic properties of the domains and their mutual stabilization, limited proteolysis was applied. Optimum conditions providing a homogeneous 10-kDa fragment at high yield were pepsin cleavage in 0.1 M NaCl/HCl pH 2.0, in the presence of 3.0 M urea. Determination of the N-terminus and the C-terminal sequence showed that cleavage occurred at the Phe88 -Arg89 peptide bond, giving rise to the complete N-terminal domain including the connecting hexapeptide. The C-terminal part of the polypeptide chain is cleaved to small fragments.Comparing the spectral properties of the isolated N-terminal domain and intact yII-crystallin proved the structure of the fragment to be closely similar to that of the native domain. Small differences in absorbance, fluorescence emission and circular dichroism point to alterations caused by the increase in surface area as a consequence of domain separation. The resistance of the 1 0-kDa fragment toward thermal and alkaline denaturation, as well as unfolding in the presence of urea or guanidine . HCl is decreased, due to the lack of domain interactions stabilizing the intact protein.Unfolding/folding kinetics of the 10-kDa fragment coincide with the second phase of the bimodal transition of intact yII-crystallin, in agreement with independent sequential folding and modular assembly of the domains within the native molecule. Domains, as distinct structural regions within globular proteins, represent autonomous cooperative folding units. As taken from their occurrence in all large proteins, 'modular assembly' [l] is a general strategy of protein folding offering long polypeptide chains the advantage of rapid self-organization. The discovery of coding and non-coding sequences in genomic DNA, and the correlation of individual protein domains with separate exons suggests 'genes-in-pieces' to imply 'proteins-in-pieces' as a common feature in the processes of transcription, splicing and translation. In certain cases, domains have been shown to originate from gene duplications. In cases where active sites reside on different domains or at domain interfaces, domain interactions may have functional significance, e.g. in terms of hinge movements, or mutual stabilization.yII-Crystallin is a typcial two-domain protein.As taken from high-resolution X-ray analysis, the N-and C-terminal halves of the molecule show twofold symmetry, each of the domains representing a sandwich consisting of two fourstrand pleated sheets organized in 'Greek key' motifs [2]. Both sequence homology and molecular topology suggest that 711-crystallin is the product of gene duplication. The high symmetry and the distinct charge pattern on the surface of the molecule may be responsible for the anomalously high stability of the molecule [3]. Whether, in this context, the domains are intrinsically stable, or whether domain interactions conCorrespondence to R. Jaenicke,