Choline-binding modules are present in some virulence factors and many other proteins of Streptococcus pneumoniae (Pneumococcus). The most extensively studied choline-binding module is C-LytA, the C-terminal moiety of the pneumococcal cell-wall amidase LytA. The three-dimensional structure of C-LytA is built up from six loop-hairpin structures forming a left-handed beta-solenoid with four choline-binding sites. The affinity of C-LytA for choline and other structural analogues allows its use as an efficient fusion tag for single-step purification of hybrid proteins. In the present study, we characterize the folding and stability of C-LytA by chemical and thermal equilibrium denaturation experiments. Unfolding experiments using guanidinium chloride at pH 7.0 and 20 degrees C suggest the existence of two partly folded states (I1 and I2) in the following model: N (native)-->I1<=>I2. The N-->I1 transition is non-co-operative and irreversible, and is significant even in the absence of a denaturant. In contrast, the I1<=>I2 transition is co-operative and reversible, with an associated freeenergy change (DeltaG(0)) of 30.9+/-0.8 kJ x mol(-1). The residual structure in the I2 state is unusually stable even in 7.4 M guanidinium chloride. Binding of choline stabilizes the structure of the native state, induces its dimerization and prevents the accumulation of the I1 species ([N]2<=>[I2]2, DeltaG(0)=50.1+/-0.8 kJ x mol(-1)). Fluorescence and CD measurements, gel-filtration chromatography and limited proteolysis suggest that I1 differs from N in the local unfolding of the N-terminal beta-hairpins, and that I2 has a residual structure in the C-terminal region. Thermal denaturation of C-LytA suggests the accumulation of at least the I1 species. These results might pave the way for an effective improvement of its biotechnological applications by protein engineering.