Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit.
The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.
Pina
David G
DG
Laboratoire trafic et signalisation, UMR 144 CNRS, Institut Curie, 75248 Paris cedex 05, France.
Gómez
Javier
J
England
Patrick
P
Craescu
Constantin T
CT
Johannes
Ludger
L
Shnyrov
Valery L
VL
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry* / Q000187:drug effects; D022641:Shiga Toxin 2 / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics; D014270:Trifluoroethanol / Q000737:chemistry* / Q000494:pharmacology
D021122:Protein Subunits; D022641:Shiga Toxin 2; C450841:shiga toxin 2 B subunit; D014270:Trifluoroethanol