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The listeriolysin O (LLO) binding and pore formation at suboptimal pH and various cholesterol concentrations in vitro are investigated. The results show that LLO activity at physiological pH is important and that its action at sites far from the cell entry may have important physiological consequences for Listeria pathogenesis.

A study was conducted to investigate the structures of three peptides corresponding to some residue of actinoporin equinatoxin II (EqTII) by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The results revealed that the 32-residue peptides lacks ordered secondary structure in water, but some residue form a helix in dodecylphosphocholine micelles.

A study is conducted on pH-induced and thermally induced unfolding of ostreolysin by means of CD, UV absorption, and intrinsic typtophan fluorescence to characterize conformational transitions associated with its functional properties. Results indicate that the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation.

Two types of model peptide is used to show that complete poly(Pro)II (PPII) CD is seen in compounds containing only one peptide bond and that the contributions of the peptide bonds on either side of the chiral alpha-carbon are distinct. The contribution from the peptide bond N-terminal to the alanine is found to correspond to the 195 nm negative maximum of the PPII spectrum, while the C-terminal peptide bond shows a broad, weak positive signal.

The effect of sodium chloride (NaCl) on the thermodynamic and conformational properties of equinatoxin (EqTxII) at pH 3.0 is studied to investigate the oligomerization of its partially unfolded states. At pH 3.0 and high salt concentrations, the heat induced and partially unfolded state of EqTxII oligomerizes and forms non-native alpha-helical secondary structure.

Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally, events associated with pore formation can modulate properties of the lipid membrane and affect its organization. Model membranes do not necessarily reproduce the physicochemical properties of the native cellular membrane, and caution is needed when transferring results from model to native lipid membranes. In this context, the utilization of novel approaches that enable studying PFTs on living cells at a single molecule level should reveal complex protein-lipid membrane interactions in greater detail. [ABSTRACT FROM AUTHOR]

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein-membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein-membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery.

Bacterial DNA gyrase is a well-established and validated target for the development of novel antibacterials. Starting from the available structural information about the binding of the natural product inhibitor, clorobiocin, we identified a novel series of 4'-methyl-N(2)-phenyl-[4,5'-bithiazole]-2,2'-diamine inhibitors of gyrase B with a low micromolar inhibitory activity by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques (differential scanning fluorimetry, surface plasmon resonance, and microscale thermophoresis). The binding mode of the potent inhibitor 18 was revealed by X-ray crystallography, confirming our initial in silico binding model. Furthermore, the high resolution of the complex structure allowed for the placement of the Gly97-Ser108 flexible loop, thus revealing its role in binding of this class of compounds. The crystal structure of the complex protein G24 and inhibitor 18 provides valuable information for further optimization of this novel class of DNA gyrase B inhibitors.