C. Ratna Prabha

Synthesis, spectroscopic characterization and DNA nuclease activity of Cu(II) complexes derived from pyrazolone based NSO-donor Schiff base ligands.

Two neutral mononuclear Cu(II) complexes have been prepared in EtOH using Schiff bases derived from 4-toluoyl pyrazolone and thiosemicarbazide. Both the ligands have been characterized on the basis of elemental analysis, IR, (1)H NMR, (13)C NMR and mass spectral data. The molecular geometry of one of these ligands has been determined by single crystal X-ray study. It reveals that these ligands exist in amine-one tautomeric form in the solid state. Microanalytical data, Cu-estimation, molar conductivity, magnetic measurements, IR, UV-Visible, FAB-Mass, TG-DTA data and ESR spectral studies were used to confirm the structures of the complexes. Electronic absorption and IR spectra of the complexes suggest a square-planar geometry around the central metal ion. The interaction of complexes with pET30a plasmid DNA was investigated by spectroscopic measurements. Results suggest that the copper complexes bind to DNA via an intercalative mode and can quench the fluorescence intensity of EB bound to DNA. The interaction between the complexes and DNA has also been investigated by agarose gel electrophoresis, interestingly, we found that the copper(II) complexes can cleave circular plasmid DNA to nicked and linear forms.

Glutamate64 to Glycine Substitution in G1 β-bulge of Ubiquitin Impairs Function and Stabilizes Structure of the Protein

Ubiquitin is a globular protein with a highly conserved sequence. Sequence conservation and compact structure make it an ideal protein for structure-function studies. One of the atypical secondary structural features found in ubiquitin is a parallel G1 beta-bulge. Glutamate at 64 is the first residue of this beta-bulge and the third residue in a type II turn. However, glycine is seen in these positions in several proteins. To understand the effects of substitution of glutamate64 by glycine on the structure, stability and function of ubiquitin, mutant UbE64G has been constructed and characterized in Saccharomyces cerevisiae. The secondary and tertiary structures of UbE64G mutant protein are only marginally different from wild-type protein (UbWt) and fluorescent form of ubiquitin (UbF45W). The earlier studies have shown that the structure and stability of UbWt and UbF45W were similar. However, UbE64G has less surface hydrophobicity than UbWt. UbE64G is found to be more stable compared with UbF45W towards guanidinium chloride induced denaturation. In vivo, complementation shows substrate proteins with Pro as the N-terminal residue, which undergo ubiquitination, have extended half-lives with UbE64G. This altered preference for Pro as opposed to Met might be related to natural preference of glutamate at 64th position in ubiquitin.

Recent advances in carbon nanotube based electrochemical biosensors

There is an increasing need for rapid, low cost, reusable, reliable and sensitive detection systems for diagnosing infectious diseases, metabolic disorders, rapidly advancing cancers and detecting the presence of environmental pollutants. Most traditional methods are invasive, slow, expensive and laborious, requiring highly specialized instruments. Introduction of biosensors with nanomaterials as transducers of signals have helped in removing the disadvantages associated with traditional detectors. The properties of high mechanical strength, better electrical conductivity and ability to serve as efficient signal transducers make carbon nanotubes (CNTs) ideal material for biosensor applications among the gamut of nanomaterials. Further, CNTs with their high surface areas, easily functionalizable surfaces for receptor immobilization are gaining importance in the construction of biosensors. The expanding field of CNTs bridges the physical sciences with biology, as chemical methods are employed to develop novel tools and platforms for understanding biological systems, in disease diagnosis and treatment. This review presents recent advances in surface functionalization of CNTs necessary for immobilization of enzymes and antibodies for biosensor applications and the methodologies used for the detection of a number of chemical and biological species. The review ends with a speculation on future prospects for CNTs in biology and medicine.

Oxidative refolding of lysozyme in trifluoroethanol (TFE) and ethylene glycol: interfering role of preexisting α-helical structure and intermolecular hydrophobic interactions

The oxidative refolding of equilibrium intermediates of lysozyme stabilized in trifluoroethanol (TFE) and ethylene glycol was monitored. Equilibrium intermediates of disulfide reduced lysozyme in TFE are known to contain considerable amounts of α‐helical structure and resemble the early intermediate in the oxidative refolding of lysozyme. We find that the intermediates in TFE do not proceed to folding; they form aggregates. However, interestingly, intermediates in ethylene glycol refold to the native state with improved folding yield. Secondary structure of these intermediates was monitored by far‐UV circular dichroism. Our results indicate that formation of α‐helical structure prior to oxidative refolding does not help the process in the case of lysozyme. Interfering with intermolecular hydrophobic interactions in the unfolded state is more productive.

Q2N and S65D Substitutions of Ubiquitin Unravel Functional Significance of the Invariant Residues Gln2 and Ser65

Ubiquitin is a small, globular protein, structure of which has been perfected and conserved through evolution to manage diverse functions in the macromolecular metabolism of eukaryotic cells. Several non-homologous proteins interact with ubiquitin through entirely different motifs. Though the roles of lysines in the multifaceted functions of ubiquitin are well documented, very little is known about the contribution of other residues. In the present study, the importance of two invariant residues, Gln2 and Ser65, have been examined by substituting them with Asn and Asp, respectively, generating single residue variants of ubiquitin UbQ2N and UbS65D. Gln2 and Ser65 form part of parallel G1 β-bulge adjacent to Lys63, a residue involved in DNA repair, cell-cycle regulated protein synthesis and imparting resistance to protein synthesis inhibitors. The secondary structure of variants is similar to that of UbF45W, a structural homologue of wild-type ubiquitin (UbWt). However, there are certain functional differences observed in terms of resistance to cycloheximide, while there are no major differences pertaining to growth under normal conditions, adherence to N-end rule and survival under heat stress. Further, expression of UbQ2N impedes protein degradation by ubiquitin fusion degradation (UFD) pathway. Such differential responses with respect to functions of ubiquitin produced by mutations may be due to interference in the interactions of ubiquitin with selected partner proteins, hint at biomedical implications.

Studies on Protein–[60]Fullerene Interactions: The Lysozyme–[60]Fullerene Model System

Abstract A lysozyme–[60]fullerene adduct was synthesized and isolated for the first time. This adduct was water‐soluble and showed strong interaction between lysozyme and [60]fullerene and was characterized by UV‐VIS and fluorescence energy transfer technique. Keeping possible biomedical applications of [60]fullerene in view, the protein–[60]fullerene interactions were studied taking lysozyme as a model protein.

Functional characterization of dosage-dependent lethal mutation of ubiquitin in Saccharomyces cerevisiae

Ubiquitin is a eukaryotic protein with 96% sequence conservation from yeast to human. Ubiquitin plays a central role in protein homeostasis and regulation of protein function. We have reported on the generation of variants of ubiquitin by in vitro evolution in Saccharomyces cerevisiae to advance our understanding of the role of the invariant amino acid residues of ubiquitin in relation to its function. One of the mutants generated, namely UbEP42, was a dosage-dependent lethal form of the ubiquitin gene, causing lethality to UBI4-deficient cells but not to ubiquitin wild-type cells. In the present study we investigated the functional reasons for the observed lethality. Expression of UbEP42 in a UBI4-deleted stress-sensitive strain resulted in an increased generation time due to a delayed S phase caused by decreased levels of Cdc28 protein kinase. Cells expressing UbEP42 displayed heightened sensitivity towards heat stress and exposure to cycloheximide. Furthermore, its expression had a negative effect on the degradation of substrates of the ubiquitin fusion degradation pathway. However, UbEP42 is incorporated into polyubiquitin chains. Collectively, our results establish that the effects seen with the mutant ubiquitin protein UbEP42 are not due to malfunction at the stage of polyubiquitination.

The ends and means of artificially induced targeted protein degradation

Studies on knockout mutants and conditional mutants are invaluable to biological research and have been used extensively to probe the intricacies of biological systems through loss of function associated with attenuation of a particular protein. Besides, RNAi technology has been developed in recent years to further aid the process of scientific inquiry. Even though, the methods, dealing with DNA and RNA have met with great success, are not without their shortcomings. In order to overcome the inadequacies of existing methods, a host of new techniques, aimed at knockdowns at the protein rather than the nucleic acid level, have been devised. Essentially, these methods can achieve rapid degradation of cellular pools of a target protein in response to an inducible signal coupled with dose-dependent modulation and exquisite temporal control, features which are absent from techniques involving manipulations at the DNA or RNA level. This review aims to provide a broad overview of a gamut of these methods, while highlighting the strengths and weaknesses of each one. Last two decades of advances presented here in the field of targeted protein degradation serve as a beacon to further research and are likely to find applications in the areas of medicine and allied fields of biology.

Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity

Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly β sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation.

Engineering degrons of yeast ornithine decarboxylase as vehicles for efficient targeted protein degradation

BACKGROUND Ornithine decarboxylase (ODC), which catalyzes the first step of polyamine biosynthesis, undergoes rapid targeted degradation (TPD) with the help of its two degron sequences, namely the N-terminal 50 residues (N50) and α/β domain (α/β) housing antizyme binding element (AzBE), in response to increased polyamine levels. Antizyme binds to AzBE of ODC and delivers it to proteasome for degradation. Entire ODC was used as a tag to demonstrate TPD of chimeric proteins. METHODS Here we fashioned three peptide sequences from yeast ODC to test their capability to act as degrons, namely N50, α/β and Nα/β (a combination of N50 and α/β), and monitored their degradation potentials in chimeric proteins. We have examined the correlation between degradation potentials and structural integrity of the peptides, to find mechanistic explanations. RESULTS Nα/β with two signals in tandem is a better degron, under the regulation of antizyme. N50 like N44 reported earlier could drive chimeric proteins to degradation, while α/β could not act as an independent degron. Strong correlation was observed between functional efficacy of the peptides and their structural integrity. N50, which was believed to be unstructured, displayed propensity for helical conformation. Nα/β exhibited optimal structure, while α/β failed to adopt native like conformation. CONCLUSIONS AND GENERAL SIGNIFICANCE Functional efficacy of the degron Nα/β is a consequence of its structural integrity. Nα/β and N50 could target chimeric proteins to degradation. However, α/β failed in the quest. Nα/β, regulated by antizyme, is better suited than N50 for TPD to understand the function of novel proteins.