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

<span style="font-size:13.0pt;mso-bidi-font-size: 8.0pt" lang="EN-GB">Q2N and E64G double mutation of ubiquitin confers a stress sensitive phenotype on <i style="mso-bidi-font-style:normal">Saccharomyces cerevisiae</i> </span>

617-620The eukaryotic protein, ubiquitin harbours a parallel β-bulge in its structure which is formed by residues Glu64(1), Ser65(2) and Gln2(X). Despite their low % frequency of occurrence in parallel β-bulges, the residues Gln2 and Glu64 have been totally conserved in ubiquitin. In a previous study, two single mutants UbQ2N and UbE64G were constructed by replacing the residues Gln2 and Glu64 with Asn and Gly, respectively to understand their importance. The choice of the residues for substitution was made on the basis of their high preference for existence in parallel β-bulge, so that the structure of mutants remains unaltered and any functional differences observed would highlight the importance of Gln2 and Glu64 in ubiquitin biology. The results from this study established that yeast cells expressing either UbQ2N or UbE64G, displayed functional differences with respect to survival upon exposure to cycloheximide and degradation of substrates by ubiquitin fusion degradation (UFD) pathway. It describes construction of the double mutant UbQ2N-E64G and its characterization. Our results showed expression of UbQ2N-E64G in stress hypersensitive SUB60 cells led to significant decrease in growth rate and prolonged half-life of substrates of UFD pathway, besides failure of complementation under heat and antibiotic stresses, providing the reason for conservation of Gln2 and Glu64 in ubiquitin sequence

Construction and functional characterization of double and triple mutants of parallel <img src='http://www.niscair.res.in/jinfo/Beta.gif' border=0>-bulge of ubiquitin

919-924Ubiquitin, a small eukaryotic protein serving as a post-translational modification on many important proteins, plays central role in cellular homeostasis and cell cycle regulation. Ubiquitin features two -bulges, the second -bulge, located at the C-terminal region of the protein along with type II turn, holds 3 residues Glu64(1), Ser65(2) and Gln2(X). Percent frequency of occurrence of such a sequence in parallel -bulge is very low. However, the sequence and structure have been conserved in ubiquitin through out the evolution. Present study involves replacement of residues in unusual -bulge of ubiquitin by introducing mutations in combination through site directed mutagenesis, generating double and triple mutants and their functional characterization. Mutant ubiquitins cloned in yeast expression vector YEp96 tested for growth profile, viability assay and heat stress complementation study have revealed significant decrease in growth rate, loss of viability and non-complementation of heat sensitive phenotype with UbE64G-S65D and UbQ2N-E64G-S65D mutations. However, UbQ2N-S65D did not show any negative effects in the above assays. Present results show that, replacement of residues in -bulge of ubiquitin exerts severe effects on growth and viability in Saccharomyces cerevisiae due to functional failure of the mutant ubiquitins UbE64G-S65D and UbQ2N-E64G-S65D

Conformational features of reduced and disulfide intact forms of hen egg white lysozyme in aqueous solution in presence of 3-chloro-1, 2-propanediol and dioxane: Implications for protein folding intermediates

97-106Conformational features of reduced and disulfide intact hen egg white lysozyme in aqueous 1,4-dioxane and 3-chloro-1, 2-propanediol solutions have been examined using circular dichroism and fluorescence spectroscopy. We find that in presence of 1, 4-dioxane, reduced lysozyme assumes a relatively compact conformational form with secondary structure closer to native state and no tertiary structure as judged by peptide and aromatic CD spectra and ANS binding studies monitored by fluorescence. Further, in presence of 40% (v/v) 3-chloro-1, 2-propanediol, disulfide intact lysozyme (DI-lysozyme) assumes a conformational form with native like secondary structure and no tertiary structure akin to a molten globule state. We correlate our results to kinetic hydrogen- deuterium exchange NMR results of the refolding of lysozyme available in literature and suggest that the conformational forms observed in our study could be models for kinetic intermediates in the refolding of lysozyme

Mutations in the ubiquitin gene of Saccharomyces cerevisiae accompanied by divergent use of CUG codon affect morphogenesis in Candida albicans

516-524UbEP42, a mutant of ubiquitin constitutes four mutations, namely S20F, A46S, L50P, and I61T. Characterization of UbEP42 and the isolated mutations revealed that UbEP42, UbL50P, and UbI61T conferred dosage-dependent lethality on S.cerevisiae, while UbS20F and UbA46S produced no effect over survival. In the present study, opportunistic humanpathogen Candida albicans has been employed here to investigate the influence of ubiquitin mutations of S. cerevisiae onmorphogenesis, besides various ubiquitin-dependent functions. The codon for leucine ‘CUG’ in S. cerevisiae is read asserine in C. albicans. Hence the protein variants expressed from the S. cerevisiae gene are added with the prefix ‘Sc’.ScUbEP42, ScUbL50P, and ScUbI61T had a negative influence over protein trafficking, lysosomal degradation of proteins,and polyubiquitination with K48 and K63 linkages in C. albicans as in S. cerevisiae. ScUbEP42 and all four derivativemutations impaired the morphogenesis of the yeast form into infective hyphal form. The impairment of morphogenesis ofC.albicans by ubiquitin mutations is unprecedented and suggests a target pathway for future therapeutics

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

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 β-bulge. Glutamate at 64 is the first residue of this β-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

Construction of hybrid solar cells exploiting the interaction of TiO2/Photosystem I complexes

567-571Proteins produce chemical energy by absorbing light in the process of photosynthesis. The use of biological photonic system has recently gained interest, especially in the field of solar energy conservation. The applications of photosynthetic components in photovoltaic material and solar cells are very promising due to their environmental compatibility and efficiency of energy conservation in photosynthesis. The recent advancement in the solar cell design includes the use of photosystem, thylakoid membrane immobilised on polymeric electrode. Previous reports have confirmed the use of PSI as photoelectric material in solar cells. The present work studies the integration of TiO2/PSI complexes with polymer electrodes. Light harvesting complexes (LHC) were isolated from spinach by homogenising cell in lysis buffer. The isolated proteins were then loaded onto DEAE-cellulose a type of ion-exchange chromatography for further purification. TiO2 layer was then spin coated onto Indium Tin Oxide coated (ITO) plates followed by dipping in a solution containing LHCs for 30 min. Upon drying, using spin coating, a thin layer of Polypyrrole electrode was casted onto it. Optical parameters and structural parameters were determined using UV-Vis analysis and XRD respectively. Efficiencies of the resulting devices were calculated under Air mass 1.5 (AM 1.5) conditions. The results establish the successful preparation of a solar cell