Acetonitrile-induced unfolding of porcine pepsin A: A proposal for a critical role of hydration structures in conformational stability.

In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.

Involvement of water in carbohydrate-protein binding.

The interactions of trimannosides 1 and 2 with Con A were studied to reveal the effects of displacement of well-ordered water molecules on the thermodynamic parameters of protein-ligand complexation. Trisaccharide 2 is a derivative of 1, in which the hydroxyl at C-2 of the central mannose unit is replaced by a hydroxyethyl moiety. Upon binding, this moiety displaces a conserved water molecule present in the Con A binding site. Structural studies by NMR spectroscopy and MD simulations showed that the two compounds have very similar solution conformational properties. MD simulations of the complexes of Con A with 1 and 2 demonstrated that the hydroxyethyl side chain of 2 can establish the same hydrogen bonds in a low energy conformation with the protein binding site as those mediated by the water molecule in the complex of 1 with Con A. Isothermal titration microcalorimetry (ITC) measurements showed that 2 has a more favorable entropy of binding compared to 1. This term, which was expected, arises from the return of the highly ordered water molecule to bulk solution. The favorable entropy term was, however, offset by a relatively large unfavorable enthalpy term. This observation was rationalized by comparing the extent of hydrogen bond and solvation changes during binding. It is proposed that an indirect interaction through a water molecule will provide a larger number of hydrogen bonds in the complex that have higher occupancies than in bulk solution, thereby stabilizing the complex.

Bactericidal action of high-power Nd:YAG laser light on Escherichia coli in saline suspension.

Infra-red light (1064 nm) from a high-power Nd:YAG laser caused more than 90% loss of viability of Escherichia coli during exposures that raised the temperature of PBS suspensions of the bacteria to 50 C in a thermocouple-equipped cuvette. In contrast, there was minimal loss of viability after heating the same suspensions to 50 degrees C in a water-bath, or in a PCR thermal cycler. The mechanism of laser killing at 50 degrees C was explored by differential scanning calorimetry, by laser treatment of transparent and turbid bacterial suspensions, and by optical absorbency studies of E. coli suspensions at 1064 nm. Taken together, the data suggested that the bactericidal action of Nd:YAG laser light at 50 degrees C was due partly to thermal heating and partly to an additional, as yet undefined, mechanism. Scanning electron microscopy revealed localized areas of surface damage on laser-exposed E. coli cells.

Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.

In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.

Microcalorimetry of chiral surfactant-cyclodextrin interactions.

The interactions of the chiral surfactants taurodeoxycholate (TDOCA) and deoxycholate (DOCA) with a range of cyclodextrins in aqueous solution have been investigated by isothermal titration microcalorimetry. In the presence of ß-cyclodextrin, the apparent critical micelle concentration (cmc) of taurodeoxycholate is increased, and the enthalpy of demicellization decreased, in a manner consistent with 1:1 complexation of TDOCA with ß-CD at low concentrations. There is no evidence for direct interaction of cyclodextrins with surfactant micelles. This is confirmed by more direct binding titrations. Below the cmc, TDOCA forms 1:1 host-guest complexes with ß-cyclodextrin (ΔH°(bind) = -32 kJ mol(-)(1), K(diss) = 0.38 mM; 25 °C, pH 7), methyl-ß-cyclodextrin (ΔH(bind) = -13 kJ mol(-)(1), K(diss) = 0.36 mM), hydroxypropyl-ß-cyclodextrin (ΔH°(bind) = -12 kJ mol(-)(1), K(diss) = 0.51 mM), and γ-cyclodextrin (ΔH°(bind) = -7.3 kJ mol(-)(1), K(diss) = 0.08 mM), but not with the smaller α-cyclodextrin. At higher cyclodextrin concentrations, the calorimetric binding data are more ambiguous, suggesting 2:1 cyclodextrin/TDOCA complexation. Similar results are found with DOCA, though experiments here are limited by the tendency of DOCA to form gels in aqueous buffers. Enhanced chromatographic or electrophoretic chiral resolution observed in mixed chiral surfactant/cyclodextrin phases could be the result of increased solubility and/or the multiplicity of chiral complexes in such systems.