RT Journal Article
T1 Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA
A1 Maestro García-Donas, María Beatriz
A1 Sanz, Jesús
AB 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 β-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 °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 (ΔG0) of 30.9±0.8 kJ·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, ΔG0=50.1±0.8 kJ·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 β-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.
PB Portland Press 
SN 0264-6021
YR 2005
FD 2005
LK https://hdl.handle.net/20.500.14352/93441.2
UL https://hdl.handle.net/20.500.14352/93441.2
LA eng
NO Beatriz MAESTRO, Jesús M. SANZ; Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA. Biochem J 15 April 2005; 387 (2): 479–488. doi: https://doi.org/10.1042/BJ20041194
NO Fundación para la Ciencia y la Tecnología 
NO Escuela Valenciana de Estudios para la Salud
DS Docta Complutense
RD 11 abr 2025