Quinaldine Red as a fluorescent probe for determining the melting temperature (Tm) of proteins: a simple, rapid and high-throughput assay.

Proteins play an important role in biological systems and several proteins are used in diagnosis, therapy, food industry etc. Thus, knowledge about the physical properties of the proteins is of utmost importance, which will aid in understanding their function and subsequent applications. The melting temperature (Tm) of a protein is one of the essential parameters which gives information about the stability of a protein under different conditions. In the present study, we have demonstrated a method for determining the Tm of proteins using the supramolecular interaction between Quinaldine Red (QR) and proteins. Using this method, we have determined the Tm of 5 proteins and compared our results with established protocols. Our results showed good agreement with the other methods and published values. The method developed in this study is inexpensive, quick, and devoid of complex instruments and pre/post-treatment of the samples. In addition, this method can be adopted for high throughput in multi-plate mode. Thus, this study projects a new methodology for Tm determination of various proteins with user friendly operation.

Biochemical characterization of clinically relevant mutations of human Translin.

DNA damage in all living cells is repaired with very high efficiency and nucleic acid binding proteins play crucial roles in repair associated processes. Translin is one such evolutionarily conserved nucleic acid interacting protein speculated to be a part of the DNA repair protein network. It is also involved in activation of RNA-induced silencing complex (RISC) along with Translin-associated factor X (TRAX) as the C3PO (component 3 promoter of RISC) complex. In the present work, we characterized ten clinically relevant variants of the human Translin protein using bioinformatic, biochemical, and biophysical tools. Bioinformatic studies using DynaMut revealed 9 out of the 10 selected mutations the Translin protein. Further analysis revealed that some mutations lead to changes in interactions with neighbouring residues in the protein structure. Using site directed mutagenesis, the point substitution variants were generated, corresponding proteins were overexpressed and purified using Ni-NTA affinity chromatography. Purified proteins form octamers similar to wild type (WT) Translin, as observed using native polyacrylamide gel electrophoresis (PAGE), gel filtration, and dynamic light-scattering (DLS) analysis. These octamers are functional and bind to single-stranded DNA (ssDNA) as well as single-stranded RNA (ssRNA) substrates. The mutant Translin proteins interact with wild type TRAX and form corresponding C3PO complexes. The C3PO complexes formed by all Translin variants with TRAX are functional in-vitro and show endoribonuclease activity. However, significant differences were observed in the extent of RNase activity in vitro. In conclusion, the clinically relevant mutations in Translin protein analysed by us exert their effect by modulating the RNase activity of the protein without altering its DNA-dependant function.

Characterization of the N- terminal domain of Mre11 protein from rice (OsMre11) Oryza sativa.

Chromosomal breaks occur in the genome of all living organisms upon exposure to ionizing radiation, xenobiotics and as intermediates during normal cell cycle progression. Most of the information on DNA repair process has emerged from bacteria, human, mice, and yeast while information on plant DNA repair genes and proteins is limited. Among other DNA repair proteins, MRE11 forms the core of the MRN (Mre11-Rad50-Nbs1) complex and is the first responder to double strand breaks (DSBs), promotes repair either by Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). Till date, MRE11 has not been biochemically characterized from plant systems. Here, we report the in vitro biochemical activities of Oryza sativa MRE11. We cloned and purified the N- terminal region of OsMre11, which represents both the nuclease and DNA binding domains. The N- terminal end of OsMre11-N protein (∼55.0 kDa) showed binding activity with dsDNA, ssDNA and G-quadruplex DNA. Tryptophan fluorescence analysis also showed that OsMre11-N protein binds to ssDNA, dsDNA and G4 DNA in a protein concentration dependant manner. Additionally, OsMre11 protein showed exonuclease activity only in the presence of Mn2+. A protein concentration dependant endonuclease activity also was observed and was enhanced in the presence of Mn2+, Mg2+ and Ca2+. Put together, OsMre11 has properties similar to its counterparts in yeast and humans and may play an important role in cellular response to DNA damage in plants, especially rice.

Role of amino acid residues important for nucleic acid binding in human Translin.

Translin is a multifunctional DNA/RNA binding protein involved in DNA repair and RNA metabolism. It has two basic regions and involvement of some residues in these regions in nucleic acid binding is established experimentally. Here we report the functional role of four residues of basic region II, Y85, R86, H88, R92 and one residue of C terminal region, K193 in nucleic acid binding using substitution mutant variants. CD analysis of the mutant proteins showed that secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Octameric state was maintained in all the mutants of basic region as evidenced by TEM, DLS, native PAGE and gel filtration analyses. However, K193G mutation completely abolished the octameric state of Translin protein and consequently its ability to bind ssDNA/ssRNA. The mutants of the basic region II exhibited a differential effect on nucleic acid binding, with R86A and R92G as most deleterious. Interestingly, H88A mutant showed higher nucleic acid binding affinity in comparison to the wild type Translin. An in silico analysis of the mutant variant sequences predicted all the mutations to be destabilizing, causing increase in flexibility and also leading to disruption of local interactions. The differential effect of mutations on DNA/RNA binding where octameric state is maintained could be attributed to these predicted disturbances.

Translin: A multifunctional protein involved in nucleic acid metabolism.

Translin, a highly conserved, DNA/RNA binding protein, is abundantly expressed in brain, testis and in certain malignancies. It was discovered initially in the quest to find proteins that bind to alternating polypurines-polypyrimidines repeats. It has been implicated to have a role in RNA metabolism (tRNA processing, RNAi, RNA transport, etc.), transcription, DNA damage response, etc. Studies from human, mice, drosophila and yeast have revealed that it forms an octameric ring, which is important for its function. Translin is a cytoplasmic protein, but under genotoxic stress, it migrates into the nucleus, binds to the break point hot spots and therefore, thought to be involved in chromosomal translocation events as well as DNA damage related response. Its structure is known and DNA binding regions, GTP binding region and regions responsible for homotypic and heterotypic interaction are known. It forms a ball like structure with open central channel for accommodating the substrate nucleic acids. Besides this, translin protein binds to 3' and 5' UTR of certain mRNAs and probably regulates their availability for translation. It is also involved in mRNA transport and cell cycle progression. It forms a heteromeric complex with translin associated factor-X (TRAX) to form C3PO complex which is involved in RNA silencing process. Recently, it has been shown that translin is upregulated under starvation conditions in Drosophila and is involved in the integration of sleep and metabolic rate of the flies. Earlier studies classified translin as a DNA repair protein; however subsequent studies showed that it is a multifunctional protein. With this background, in this review we have summarized the translin biochemical activities, cellular function as well as structural properties of this important protein.