Interaction of bovine serum albumin (BSA) with two series of dipolar molecules having both rigid and flexible structures has been studied by monitoring the spectral and temporal behavior of the intramolecular charge transfer fluorescence of the systems. The binding sites of the molecular systems in BSA have been located with the help of docking studies. Three different sites of varying hydrophobicity have been identified where these molecules are located. Binding in the hydrophobic domains of BSA leads to a blue shift of the fluorescence spectra and an enhancement of fluorescence intensity and lifetime. This enhancement is found to be the largest for flexible systems in which internal motion serves as a nonradiative decay route. In the BSA-bound condition, some of the dipolar molecules exhibit not-so-common "dip-rise-dip" time-resolved fluorescence anisotropy profiles. It is shown that a large difference of the fluorescence lifetimes of the protein-bound and unbound molecules is one of the factors that contributes to this kind of anisotropy profiles. As internal motion is often responsible for the short fluorescence lifetime of the flexible dipolar molecules, a large increase in the fluorescence lifetime of these systems occurs if binding to BSA leads to disruption/prevention of this motion. It thus appears that it might be possible to obtain information on the prevention/disruption of nonradiative pathway on protein binding from the anisotropy profiles of the kind discussed above. However, since the present study reveals cases where a large change in fluorescence lifetime also occurs due to other reasons, one needs to be careful prior to making any conclusion.

The human spike protein sequences from Asia, Africa, Europe, North America, South America, and Oceania were analyzed by comparing with the reference severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) protein sequence from Wuhan‐Hu‐1, China. Out of 10333 spike protein sequences analyzed, 8155 proteins comprised one or more mutations. A total of 9654 mutations were observed that correspond to 400 distinct mutation sites. The receptor binding domain (RBD) which is involved in the interactions with human angiotensin‐converting enzyme‐2 (ACE‐2) receptor and causes infection leading to the COVID‐19 disease comprised 44 mutations that included residues within 3.2 Å interacting distance from the ACE‐2 receptor. The mutations observed in the spike proteins are discussed in the context of their distribution according to the geographical locations, mutation sites, mutation types, distribution of the number of mutations at the mutation sites and mutations at the glycosylation sites. The density of mutations in different regions of the spike protein sequence and location of the mutations in protein three‐dimensional structure corresponding to the RBD are discussed. The mutations identified in the present work are important considerations for antibody, vaccine, and drug development.

The pleckstrin homology (PH) domain is a conserved module present in many signal transducing and cytoskeletal proteins. Here we report the 2.8 A crystal structure of the PH domain from dynamin. This domain consists of seven beta-strands forming two roughly orthogonal antiparallel beta-sheets terminating with an amphipathic alpha-helix. The structure also reveals a non-covalent dimeric association of the PH domain and a hydrophobic pocket surrounded by a charged rim. The dynamin PH domain structure is discussed in relation to its potential role in mediating interactions between proteins.

<p>The human SARS-CoV-2 spike protein sequences from Asia, Africa, Europe, North America, South America and Oceania were analyzed by comparing with the reference SARS-CoV-2 protein sequence from Wuhan-Hu-1, China. Out of 10,333 spike protein sequences analyzed, 8,155 proteins comprised one or more mutations. A total of 9,654 mutations were observed that correspond to 400 distinct mutation sites. The receptor binding domain (RBD) which is involved in the interactions with human ACE-2 receptor and causes infection leading to the COVID-19 disease comprised 44 mutations that included residues within 3.2 Å interacting distance from the ACE-2 receptor. The mutations observed in the spike proteins are discussed in the context of their distribution according to the geographical locations, mutation sites, mutation types, distribution of the number of mutations at the mutation sites and mutations at the glycosylation sites. The density of mutations in different regions of the spike protein sequence and location of the mutations in protein three-dimensional structure corresponding to the RBD are discussed. The mutations identified in the present work are important considerations for antibody, vaccine and drug development.</p>

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition.Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.

Background: SpChiD, a family 18 glycosyl hydrolase, transglycosylates chitooligosaccharides. Results: Transglycosylation of SpChiD was improved in terms of the quantity of TG products produced and the extended duration of TG. Conclusion:The new findings unravel possibilities of modulating chitinases for improved transglycosylation. Significance: Variants of SpChiD can be used to develop a bio-process for large scale production of longer chain chitooligosaccharides.

Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC1551 strains of the Mycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acid-residue common region in 22 proteins. The pairwise sequence identities were as low as 18%. Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region. The secondary structure corresponding to this region is predicted to be a mixture of a-helices and b-strands. Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function. We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs. This region is preceded by a hydrophobic region, comprising 40-100 amino acid residues, that is flanked by charged amino acid residues. Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions. Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41-43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980). These correspond to the AB repeats that were first identified in some proteins of the Methanosarcina mazei genome, and were demonstrated as surface antigens. We observed the AB repeats also in several other proteins of hitherto uncharacterized function in Archaea and Bacteria genomes. Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues. The secondary structure corresponding to the AB repeat is predicted mainly as 4 b-strands. We suggest that proteins with AB repeats in Mycobacterium tuberculosis and other genomes may be associated as surface antigens. The M. leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in the M. leprae genome compared to the M. tuberculosis genome.

Resistin, a cysteine-rich adipocytokine, proposed as a link between obesity and diabetes in mice, was shown as a proinflammatory molecule in humans. We earlier reported that human resistin (hRes), a trimer, was resistant to heat and urea denaturation, existed in an oligomeric polydispersed state, and showed a concentrationdependent conformational change. These properties and an intimate correlation of hRes expression with cellular stress prompted us to investigate hRes as a possible chaperone. Here, we show that recombinant human resistin was able to protect the heat-labile enzymes citrate synthase and Nde1 from thermal aggregation and inactivation and was able to refold and restore their enzymatic activities after heat/guanidinium chloride denaturation. Furthermore, recombinant human resistin could bind misfolded proteins only. Molecular dynamics-based association-dissociation kinetics of hRes subunits pointed to resistin being a molecular chaperone. Bis-ANS, which blocks surface hydrophobicity, abrogated the chaperone activity of hRes, establishing the importance of surface hydrophobicity for chaperone activity. Replacement of Phe49 with Tyr (F49YhRes), a critical residue within the hydrophobic patch of hRes, although it could prevent thermal aggregation of citrate synthase and Nde1, was unable to refold and restore their activities. Treatment of U937 cells with tunicamycin/thapsigargin resulted in reduced hRes secretion and concomitant localization in the endoplasmic reticulum. Escherichia coli transformants expressing hRes could be rescued from thermal stress, pointing to hRes's chaperone-like function in vivo. HeLa cells transfected with hRes showed protection from thapsigargin-induced apoptosis. In conclusion, hRes, an inflammatory protein, additionally exhibited chaperone-like properties, suggesting a possible link between inflammation and cellular stress.protein folding | chaperokine R esistin, a small cysteine-rich secreted protein, is predominantly produced in human macrophages (1, 2). Resistin levels in human serum could neither be associated with obesity nor linked with insulin resistance (3), pointing to possible other role(s) for this hormone. We, and later others, showed that human resistin (hRes) is a proinflammatory molecule that stimulates the synthesis and secretion of TNF-α and IL-12 from macrophages through an NF-κB-activated pathway (4, 5). hRes mRNA levels are strongly induced by TNF-α and IL-6 in human peripheral blood mononuclear cells (6, 7). Although human and mouse resistin share 64.4 and 59% sequence homology at mRNA and amino acids levels, respectively, they differ considerably in terms of their structural organization (8). We earlier reported, based on extensive biophysical analyses, that recombinant human resistin (rhRes) is a highly stable molecule that exists in oligomeric states as a function of concentration with no major loss in helicity and displays slightly altered tertiary structure with an increase in temperature (9, 10). The variable oligomeric states and poly-dispersity...