Cys mutants as tools to study the oligomerization of the pore-forming toxin sticholysin I.

Sticholysin I (StI) is a water-soluble protein with the ability to bind membranes where it oligomerizes and forms pores leading to cell death. Understanding the assembly property of this protein may be valuable for designing potential biotechnological tools, such as stable or structurally defined nanopores. In order to get insights into the stabilization of StI oligomers by disulfide bonds, we designed and characterized single and double cysteine mutants at the oligomerization interface.

Architecture of the pore forming toxin sticholysin I in membranes.

Sticholysin I (StI) is a toxin produced by the sea anemone Stichodactyla helianthus and belonging to the actinoporins family. Upon binding to sphingomyelin-containing membranes StI forms oligomeric pores, thereby leading to cell death. According to recent controversial experimental evidences, the pore architecture of actinoporins is a debated topic.

A commonly used spin label: S-(2,2,5,5-tetramethyl-1-oxyl-delta3-pyrrolin-3-ylmethyl) methanethiosulfonate.

The title compound, C(10)H(18)NO(3)S(2), which finds application as a spin label, has triclinic (P\overline{1}) symmetry at 100 (2) K with two independent molecules in the asymmetric unit. Both molecules are very similar with respect to bond lengths and angles, but molecule 2 shows disordering of its side chain. The pyrroline rings differ slightly with respect to the position of the NO group, which in both cases are sterically shielded by the surrounding methyl groups.

Molecular mechanisms of gene regulation studied by site-directed spin labeling.

The technique of site-directed spin labeling using cysteine substitution mutagenesis followed by modification of the sulfhydryl group with a nitroxide reagent is emerging as a valuable alternative for the determination of protein folds and conformational changes in a variety of systems. The incorporation of pairs of nitroxides allows determination of intramolecular distances and distance changes with a spatial resolution at the level of the backbone fold under conditions relevant to function. The methodology of electron paramagnetic resonance spectral data acquisition and interpretation is reviewed with studies on conformational changes of Tet repressor (TetR) and the human immunodeficiency virus type 1 reverse transcriptase (RT) on interaction with nucleic acid substrates or inhibitors in solution.