Dethiolation of protein mixed-disulfides

The dithiol proteins, glutaredoxin, thioredoxin, and protein disulfide isomerase, were examined as dethiolases (i.e., reductases for protein mixed-disulfides) by studying the specificity and reactivity for an S-glutathiolated protein mixture. The 35S-glutathiolated protein mixture was prepared from 35S-labeled rat hepatocytes by diamide treatment. Dethiolation of individual 35S-labeled proteins was analyzed by combining SDS-PAGE and autoradiography. The dithiol proteins greatly enhanced dethiolation rates and could completely dethiolate all of the S-glutathiolated proteins. The dethiolation rate for individual proteins by each dithiol protein was compared and glutaredoxin was the most effective for every S-glutathiolated hepatocyte protein. When testing the reduction of insulin disulfide, we found that glutaredoxin did not catalyze insulin reduction, while protein disulfide isomerase and thioredoxin did. This suggests that glutaredoxin may be specific for S-glutathiolated proteins. The redox potential of glutaredoxin was determined to be -0.159 ± 0.004 V, indicating that glutaredoxin was thermodynamically a weaker reductant than E. coli thioredoxin and similar to protein disulfide isomerase. The effective dethiolation by glutaredoxin compared to thioredoxin and protein disulfide isomerase, therefore, might be determined by kinetic factors. Glutathione-binding at the active site of glutaredoxin was suggested as an important factor for the effective dethiolation. The results suggested that glutaredoxin might be the major dethiolase for S-glutathiolated proteins in cells;Dethiolation of the mixture of 35S-glutathiolated hepatocyte proteins by glutathione was also studied. All the 35S-glutathiolated proteins were dethiolated by glutathione. Dethiolation of either the mixed hepatocyte proteins or pure S-glutathiolated phosphorylase b was saturable with respect to the concentration of glutathione. The half maximal rate of dethiolation occurred at 0.16 mM GSH. The dethiolation rate decreased in the presence of denaturing agents such as guanidinium chloride and sodium dodecyl sulfate, as well as the glutathione analogs, S-methylglutathione and glutathione sulfate. The saturation kinetics, inhibition by glutathione analogs and protein denaturants, strongly suggest that glutathione forms noncovalent complexes with S-thiolated proteins during dethiolation. This glutathione-binding suggests that liver proteins may contain glutathione-binding sites closely associated with the reactive sulfhydryls that undergo S-thiolation and dethiolation

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a “Cascade” ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called “interference.” After target recogni- tion by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mecha- nism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD- nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mecha- nism for 3′ to 5′ nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3′ to 5′ trans- location of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process

Dethiolation of protein mixed-disulfides

The dithiol proteins, glutaredoxin, thioredoxin, and protein disulfide isomerase, were examined as dethiolases (i.e., reductases for protein mixed-disulfides) by studying the specificity and reactivity for an S-glutathiolated protein mixture. The 35S-glutathiolated protein mixture was prepared from 35S-labeled rat hepatocytes by diamide treatment. Dethiolation of individual 35S-labeled proteins was analyzed by combining SDS-PAGE and autoradiography. The dithiol proteins greatly enhanced dethiolation rates and could completely dethiolate all of the S-glutathiolated proteins. The dethiolation rate for individual proteins by each dithiol protein was compared and glutaredoxin was the most effective for every S-glutathiolated hepatocyte protein. When testing the reduction of insulin disulfide, we found that glutaredoxin did not catalyze insulin reduction, while protein disulfide isomerase and thioredoxin did. This suggests that glutaredoxin may be specific for S-glutathiolated proteins. The redox potential of glutaredoxin was determined to be -0.159 ± 0.004 V, indicating that glutaredoxin was thermodynamically a weaker reductant than E. coli thioredoxin and similar to protein disulfide isomerase. The effective dethiolation by glutaredoxin compared to thioredoxin and protein disulfide isomerase, therefore, might be determined by kinetic factors. Glutathione-binding at the active site of glutaredoxin was suggested as an important factor for the effective dethiolation. The results suggested that glutaredoxin might be the major dethiolase for S-glutathiolated proteins in cells;Dethiolation of the mixture of 35S-glutathiolated hepatocyte proteins by glutathione was also studied. All the 35S-glutathiolated proteins were dethiolated by glutathione. Dethiolation of either the mixed hepatocyte proteins or pure S-glutathiolated phosphorylase b was saturable with respect to the concentration of glutathione. The half maximal rate of dethiolation occurred at 0.16 mM GSH. The dethiolation rate decreased in the presence of denaturing agents such as guanidinium chloride and sodium dodecyl sulfate, as well as the glutathione analogs, S-methylglutathione and glutathione sulfate. The saturation kinetics, inhibition by glutathione analogs and protein denaturants, strongly suggest that glutathione forms noncovalent complexes with S-thiolated proteins during dethiolation. This glutathione-binding suggests that liver proteins may contain glutathione-binding sites closely associated with the reactive sulfhydryls that undergo S-thiolation and dethiolation.</p

A Fast Performance Analysis Tool for Multicore Multithreaded Communication Processors.IEEE Computer Society ,2008,Volume 00:135

Abstract To allow fast communication processor (CP

Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein

The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins

D-ribose-5-phosphate isomerase from spinach: heterologous1

A cDNA encoding spinach chloroplastic ribose-5-phosphate isomerase (RPI) was cloned and overexpressed in Escherichia coli, and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from spinach leaves (A. C. Rutner, 1970, Biochemistry 9, 178 -184). Phosphate, used as a buffer in previous studies, is a competitive inhibitor of RPI with a K i of 7.9 mM. D-Arabinose 5-phosphate is an effective inhibitor, while Dxylulose-5 phosphate is not, indicating that the configuration at carbon-3 contributes to substrate recognition. Although D-arabinose 5-phosphate binds to RPI, it is not isomerized, demonstrating that the configuration at carbon-2 is crucial for catalysis. Alignment of RPI sequences from diverse sources showed that only 11 charged amino acid residues of the 236-residue subunit are conserved. The possible function of four of these residues was examined by site-directed mutagenesis. As the catalyst for the interconversion of D-ribose 5-phosphate and D-ribulose 5-phosphate, RPI 5 (EC 5.3.1.6), plays an essential role in the Calvin cycle of photosynthesis and in the oxidative pentose phosphate pathway of both photosynthetic and nonphotosynthetic organisms (1). RPI, in concert with ribulose-5-phosphate epimerase, facilitates partitioning of pentose phosphates between these two pathways in photosynthetic organisms, depending on metabolic needs and the redox status of cells. D-Ribose 5-phosphate itself is the substrate for the synthesis of phosphoribosyl pyrophosphate, which serves as a precursor for histidine, tryptophan, and nucleotides (2), and D-ribulose 5-phosphate in turn is a precursor for riboflavin (3)