Deciphering protein microenvironment by using a cysteine specific switch-ON fluorescent probe.

Fluorescent probes provide an unparalleled opportunity to visualize and quantify dynamic events. Here, we employ a medium-size, cysteine specific coumarin based switch-ON fluorescent probe 'L' to track protein unfolding profiles and accessibility of cysteine residues in proteins. It was established that 'L' is highly selective and exhibits no artifact due to interaction with other bystander species. 'L' is able to gauge subtle changes in protein microenvironment and proved to be effective in delineating early unfolding events that are difficult to otherwise discern by classic techniques such as circular dichroism. By solving the X-ray structure of TadA and probing the temperature dependent fluorescence-ON response with native TadA and its cysteine mutants, it was revealed that unfolding occurs in a stage-wise manner and the regions that are functionally important form compact sub-domains and unfold at later stages. Our results assert that probe 'L' serves as an efficient tool to monitor subtle changes in protein structure and can be employed as a generic dye to study processes such as protein unfolding.

Rassf Proteins as Modulators of Mst1 Kinase Activity.

Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway.