Browsing by Subject "Proteins--Analysis."

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Open Access
Hanein-1, a novel conserved eukaryotic protein ubiquitously expressed in human tissues
(2008) Erkek, Serap
HANEIN-1 was first identified in a study investigating 3’ transcriptional regulatory elements of the Na+ /I- symporter gene. The protein is highly conserved among eukaryotes and bears no domain similarities with any known proteins. In databases, it is described as coiled coil containing-124 hypothetical protein and predicted to encode a 223 amino acid-protein. In this project, our aim was to characterize this highly conserved protein by using several bioinformatics and biochemical methodologies. Sequence similarity search analysis showed that it had around 75% identity in mammals, 50% identity with insects and nematodes. Expressional analysis revealed that HANEIN-1 was expressed in all tissues ubiquitously with a remarkable expression status in skeletal muscle. Beside providing information about expression status of HANEIN-1, northern blotting showed that HANEIN-1 transcript size was approximately 1000 bp. Regarding protein level expression, western blotting revealed that HANEIN-1 encoded a 33 kDa protein and protein stability was affected in a different way upon labeling with Flag epitope at N-ter and C-ter. Yeast double hybrid screening performed in our laboratory showed that HANEIN-1 interacted with RASGEF1B, which was a guanine nucleotide exchange factor not fully characterized. Expressional analysis displayed that RASGEF1B expression profile inversely correlated with HANEIN-1. Finally, serine-scanning mutagenesis analysis showed that site-directed mutagenesis of serine at position 194 significantly affected the stability of the protein.
Open Access
Nitroolefin functionalized bodipy dyes for protein labeling
(2013) Turgut, Hatice
Protein labeling has significant importance in terms of visualizing dynamics of proteins, cell-cell interactions, mechanisms of life cycles of proteins, etc. Proteins are labeled by either synthetic or natural molecules with purposes such as analysis of 3D structures, determination of turnover number, covalent modifications and tracking protein-protein interactions. In addition to this, sensing and signalling thiol groups have gained popularity recently. Nitroolefin groups on dyes are good Micheal acceptors which undergo fast and selective reaction with thiol moieties. With this knowledge, in this study, we aimed to obtain derivatives of BODIPY dyes having nitroolefin substituents on its different positions. Nitroolefin functionalization of BODIPY dyes was targeted to result in conjugation of nitroolefins with thiol groups such as those belonging to cysteine residues on proteins. Three different nitroolefin functionalized BODIPY dyes have been designed, synthesized and characterized successfully. Incorporating triethylene glycol (TEG) units onto BODIPYs increased water-solubility of the molecules. To prove bioconjugation of the dyes with proteins, absorbance and emission changes were recorded after reaction with both L-cysteine and Bovine Serum Albumin (BSA) and large spectral changes were obtained. The result suggests that nitroolefin functionalization of BODIPY dyes is a promising way to sense biological thiols and hence labeling proteins having thiol groups.