Effect of site-directed point mutations on protein misfolding: A simulation study

A Monte Carlo simulation based sequence design method is proposed to investigate the role of site-directed point mutations in protein misfolding. Site-directed point mutations are incorporated in the designed sequences of selected proteins. While most mutated sequences correctly fold to their native conformation, some of them stabilize in other nonnative conformations and thus misfold/unfold. The results suggest that a critical number of hydrophobic amino acid residues must be present in the core of the correctly folded proteins, whereas proteins misfold/unfold if this number of hydrophobic residues falls below the critical limit. A protein can accommodate only a particular number of hydrophobic residues at the surface, provided a large number of hydrophilic residues are present at the surface and critical hydrophobicity of the core is preserved. Some surface sites are observed to be equally sensitive toward site-directed point mutations as the core sites. Point mutations with highly polar and charged amino acids increases the misfold/unfold propensity of proteins. Substitution of natural amino acids at sites with different number of nonbonded contacts suggests that both amino acid identity and its respective site-specificity determine the stability of a protein. A clash-match method is developed to calculate the number of matching and clashing interactions in the mutated protein sequences. While misfolded/unfolded sequences have a higher number of clashing and a lower number of matching interactions, the correctly folded sequences have a lower number of clashing and a higher number of matching interactions. These results are valid for different SCOP classes of proteins.     

Role of sulphated tyrosine residue in influencing the biologic activity of human cholecystokinin-33

To evaluate the role of the sulphated tyrosine residue in position 27 in human cholecystokinin-33, parallel bioassay of the sulphated form of human cholecystokinin-33 and the unsulphated form of human cholecystokinin-33 was performed on the pancreatic protein secretion. Both peptides increased the protein output in a dose-related manner. However, the sulphated form possessed a considerably higher activity than the sulphated form. The relative potency of the unsulphated human cholecystokinin-33 compared to that of the sulphated human cholecystokinin-33 (taken as 1.0) was 0.08. From the results, it was suggested that the sulphated tyrosine may play an important role in controlling the activity of the longer molecular forms as well as that of the smaller forms of cholecystokinin.     

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.     

Expression of gonococcal transferrin-binding protein 1 causes Escherichia coli to bind human transferrin.

The gene for gonococcal transferrin-binding protein 1 (TBP1) was cloned behind an inducible promoter in Escherichia coli. The resultant strain was capable of binding human transferrin with the same specificity as that of the gonococcus. E. coli expressing TBP1 did not internalize transferrin-bound iron or grow on transferrin as a sole iron source.     

Murine splenocyte proliferation by porin of Shigella dysenteriae type 1 and inhibition of bacterial invasion of HeLa cell by anti-porin antibody

Abstract A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.     

Proteome Analysis Database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes

The SWISS-PROT group at EBI has developed the Proteome Analysis Database utilising existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes (http://www.ebi.ac. uk/proteome/). The two main projects used, InterPro and CluSTr, give a new perspective on families, domains and sites and cover 31-67% (InterPro statistics) of the proteins from each of the complete genomes. CluSTr covers the three complete eukaryotic genomes and the incomplete human genome data. The Proteome Analysis Database is accompanied by a program that has been designed to carry out InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.     

Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.     

Influence of potassium solubilizing microorganism (Bacillus mucilaginosus) and waste mica on potassium uptake dynamics by sudan grass (Sorghum vulgare Pers.) grown under two Alfisols

The main aim of this research was to study the dynamics of K release from waste mica inoculated with potassium solubilizing microorganism (Bacillus mucilaginosus) and to investigate its effectiveness as potassic-fertilizer using sudan grass (Sorghum vulgare Pers.) var Sudanensis as test crop grown under two Alfisols. Results revealed that application of mica significantly enhanced biomass yield, uptake and per cent K recoveries by sudan grass than control (no-K). Biomass yield, uptake and per cent K recoveries increased further when mica was inoculated with bacterial strain in both the soils than uninoculated mica. Alfisol from Hazaribag recorded higher yield, uptake and K recoveries than Alfisol from Bhubaneswar. The dynamics of K in soils indicated that K was released from mica to water-soluble and exchangeable pools of K due to inoculation of mica with Bacillus mucilaginosus in both the soils. Significantly greater amounts of water-soluble, exchangeable and non-exchangeable K were maintained in Alfisol from Hazaribag than Bhubaneswar. Release kinetics of K showed significant release of K from mica treated with bacterial strain. Significant correlation between biomass yield, K uptake by sudan grass and different pools of K in soils were observed. X-ray diffraction analysis indicates greater dissolution of mica due to inoculation of Bacillus mucilaginosus strain in both the soils. Thus, bio-intervention of waste mica could be an alternative and viable technology to solubilize insoluble K into plant available pool and used efficiently as a source of K-fertilizer for sustaining crop production and maintaining soil potassium.