Structural basis of conformational variance in phosphorylated and non-phosphorylated states of PKCßII.

PKCßII activation is achieved by primary phosphorylation at three phosphorylation sites, followed by the addition of secondary messengers for full activation. Phosphorylation is essential for enzyme maturation, and the associated conformational changes are known to modulate the enzyme activation. To probe into the structural basis of conformational changes on phosphorylation of PKCßII, a comprehensive study of the changes in its complexes with ATP and ruboxistaurin was performed. ATP is a phosphorylating agent in its phosphorylation reaction, and ruboxistaurin is its specific inhibitor. This study provides insight into the differences in the important structural features in phosphorylated and non-phosphorylated states of PKCßII. Less conformational changes when PKCßII is bound to inhibitor in comparison to when it is bound to its phosphorylating agent in both states were observed. The interactions of ruboxistaurin significant in restricting PKCßII to attain the conformational state competent for full activation are reported.

Composition and temperature-induced structural changes in lead-tellurite glasses on different length scales.

Processes occurring at macroscopic and microscopic length scales across the glass transition (T(g)) in lead-tellurite glass (PbO)(x)(TeO(2))(1-x) (x = 0.1-0.3) are investigated using Brillouin and Raman spectroscopy, respectively. For all the samples, the temperature dependence of the longitudinal acoustic (LA) mode is found to exhibit a universal scaling below T(g) and a rapid softening above T(g). The lower value of elastic modulus at a higher concentration of network modifier PbO, estimated from Brillouin data, arises due to loss of network rigidity. From quantitative analysis of the reduced Raman spectra, several modes are found to exhibit anomalous changes across T(g). Instead of the expected anharmonic behaviour, several modes exhibit hardening, suggesting stiffening of the stretching force constants with temperature, the effect being more pronounced in glasses with higher x. In addition, incorporation of PbO in the glass is also found to narrow down the bond-length distribution, as evident from the sharpening of the Raman bands. The stiffening of the force constants of molecular units at a microscopic length scale and the decrease of elastic constant attributed to loss of network rigidity on a macroscopic length scale appear to be opposite. These different behaviours at two length scales are understood on the basis of a microscopic model involving TeO(n) and PbO units in the structure.

Microscopic analysis of fluorescence resonance energy transfer (FRET).

We describe here detailed practical procedures for implementing various approaches of fluorescence resonance energy transfer (FRET) in microscope-based measurements. A comprehensive theoretical formalism is developed and the different experimental procedures are outlined. A step-by-step protocol is provided for preparing the specimens for FRET measurements, data acquisition procedures, analysis, and quantification. Particular emphasis is given to exemplify the FRET applications in the study of protein-protein interactions.