Developing a circularly permuted variant of Renilla luciferase as a bioluminescent sensor for measuring Caspase-9 activity in the cell-free and cell-based systems.

Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.

Remarkable improvement of methylglyoxal synthase thermostability by His-His interaction.

Lately it has been proposed that interaction between two positively charged side chains can stabilize the folded state of proteins. To further explore this point, we studied the effect of histidine-histidine interactions on thermostability of methylglyoxal synthase from Thermus sp. GH5 (TMGS). The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close distance and form a surface loop. Here, two modified enzymes were produced by site-directed mutagenesis (SDM); one of them, one histidine (TMGS-HH(O)), and another two histidines (TMGS-HHH(O)) were inserted between Arg22 and His23 (H(O)). In comparison with the wild type, TMGS-HH(O) thermostability increased remarkably, whereas TMGS-HHH(O) was very unstable. To explore the role of His23 in the observed phenomenon, the original His23 in TMGS-HHH(O) was replaced with Ala (TMGS-HHA). Our data showed that the half-life of TMGS-HHA decreased in relation to the wild type. However, its half-life increased in comparison with TMGS-HHH(O). These results demonstrated that histidine-histidine interactions at position 23 in TMGS-HH(O) probably have the main role in TMGS thermostability.

Transmitting the allosteric signal in methylglyoxal synthase.

The homohexameric enzyme methylglyoxal synthase (MGS) converts dihydroxyacetone phosphate (DHAP) to methylglyoxal and phosphate. This enzyme is allosterically inhibited by phosphate. The allosteric signal induced by phosphate in MGS from Thermus sp. GH5 (TMGS) has been tracked by site-directed mutagenesis, from the binding site of phosphate to the pathways that transmit the signal, and finally to the active site which is the receiver of the signal. In TMGS, Ser-55 distinguishes the inhibitory phosphate from the phosphoryl group of the substrate, DHAP, and transmits the allosteric signal through Pro-82, Arg-97 and Val-101 to the active site. Furthermore, the addition of a C-terminal tail to TMGS reinforces the allosteric signal by introducing a new salt bridge between Asp-10 and an Arg in this tail. Lastly, the active site amino acid, Gly-56, is shown to be involved in both allostery and phosphate elimination step from DHAP by TMGS. Interestingly, some of the mutations also trigger homotropic allostery, supporting the hypothesis that allostery is an intrinsic property of all dynamic proteins. The details of the TMGS allosteric network discussed in this study can serve as a model system for understanding the enigmatic allosteric mechanism of other proteins.

Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase.

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3´-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus (TMGS(+)). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and TMGS(+), two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway.

Conferral of allostery to Thermus sp. GH5 methylglyoxal synthase by a single mutation.

There is huge number of oligomeric proteins that show allosteric behaviour as soon as their allosteric effector is provided. Thermus sp. GH5 methylglyoxal synthase is also a homohexameric protein, which displays cooperative behaviour when phosphate concentration increases. Previous studies on this enzyme have indicated that binding of phosphate leads to formation of specific interactions which makes the enzyme capable of displaying allosteric behaviour when its substrate is bound. In this study, it has been shown that a single mutation, independent of phosphate, provides the requirements for showing such cooperative behaviour. However, it is proposed that the allosteric mechanism triggered by phosphate is different from that applied by the mutation. These findings point towards the fact that allostery can be acquired, modulated or eliminated by any alteration in structure and/or dynamics of the proteins and all proteins are potentially capable of showing cooperative behaviour as soon as their prerequisites for this phenomenon are provided by the allosteric effector.