Mutagenic Primers Research Articles

Mutagenic Primer Design for Mismatch PCR-RFLP SNP Genotyping Using a Genetic Algorithm

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is useful in small-scale basic research studies of complex genetic diseases that are associated with single nucleotide polymorphism (SNP). Designing a feasible primer pair is an important work before performing PCR-RFLP for SNP genotyping. However, in many cases, restriction enzymes to discriminate the target SNP resulting in the primer design is not applicable. A mutagenic primer is introduced to solve this problem. GA-based Mismatch PCR-RFLP Primers Design (GAMPD) provides a method that uses a genetic algorithm to search for optimal mutagenic primers and available restriction enzymes from REBASE. In order to improve the efficiency of the proposed method, a mutagenic matrix is employed to judge whether a hypothetical mutagenic primer can discriminate the target SNP by digestion with available restriction enzymes. The available restriction enzymes for the target SNP are mined by the updated core of SNP-RFLPing. GAMPD has been used to simulate the SNPs in the human SLC6A4 gene under different parameter settings and compared with SNP Cutter for mismatch PCR-RFLP primer design. The in silico simulation of the proposed GAMPD program showed that it designs mismatch PCR-RFLP primers. The GAMPD program is implemented in JAVA and is freely available at http://bio.kuas.edu.tw/gampd/.

RhoGDI SUMOylation at Lys-138 Increases Its Binding Activity to Rho GTPase and Its Inhibiting Cancer Cell Motility

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

Prenatal screening for β-thalassemia major reveals new and rare mutations in the Pakistani population

β-Thalassemia is the most common genetic disorder in Pakistan, where more than 6000 affected children are born annually, and the carrier population is around 10 million. The objective was to study β-globin gene mutations in chorionic villous biopsy samples. Prenatal screening of 383 pregnant women between 2003 and 2010 was carried out using a panel of 13 mutation primers and amplification refractory mutations system (ARMS)-PCR. In addition, DNA sequencing was used to confirm uncharacterized mutations and in some cases fetal disease status was confirmed by linkage analysis. Families enrolled in this study represented major ethnic groups in Pakistan. Of the 13 mutations tested, three mutations accounted 71% of the total, including IVS1-5(G-C)[HBB:c.92+5G>C], codon 8/9(+G) [HBB:c.27_28insG] and del 619[NG_000007.3:g71609-72227del619]. Mutations in four uncharacterized samples were later confirmed by DNA sequencing as -88(C-T)[HBB:c.-138C>G], -90(C-T)[HBB:c.-140C>T] and codon 59(+T)[HBB:c.178_179insT]. To our knowledge, this is the first report of these mutations in Pakistan. Moreover, 19.2% fetal samples were normal and 52.3% heterozygous, whereas 26.4% were affected with thalassemia major. IVS1-5:IVS1-5 was the most common genotype in fetal samples. Prenatal diagnosis of β-thalassemia using ARMS PCR is an efficient approach for reducing the burden of this disease in Pakistan. In addition, rare mutations reported in this study should be incorporated in the diagnostic strategy.

Acute megakaryoblastic leukemia and severe pulmonary fibrosis in a child with down syndrome: Successful treatment with ultra low-dose cytarabine using GATA1 mutation to monitor minimal residual disease

Acute megakaryoblastic leukemia and severe pulmonary fibrosis in a child with down syndrome: Successful treatment with ultra low-dose cytarabine using GATA1 mutation to monitor minimal residual disease

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

Mapping Structural Determinants within Third Intracellular Loop That Direct Signaling Specificity of Type 1 Corticotropin-releasing Hormone Receptor

The type 1 corticotropin-releasing hormone receptor (CRH-R1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg(292)-Met(295) and Lys(311)-Lys(314) reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75% and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg(292), Lys(297), Arg(310), Lys(311), and Lys(314) as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg(299) reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G(q) proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

Phosphorylation of PhoP Protein Plays Direct Regulatory Role in Lipid Biosynthesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis PhoP is essential for virulence and intracellular growth of the tubercle bacilli. Genetic evidence suggests that PhoP regulates complex lipid biosynthesis, and absence of some of these lipid molecules in a phoP mutant partly accounts for its attenuated growth in macrophages and/or mice. To investigate the mechanism of regulation, here we demonstrate the essentiality of phosphorylation of PhoP in the regulation of complex lipid biosynthesis. We show that phosphorylated PhoP activates transcription of pks2 and msl3, gene(s) encoding polyketide β-ketoacyl synthases through direct DNA binding at the upstream regulatory region(s) of the target genes. Our results identify the genetic determinants recognized by PhoP and show that activation of target genes requires interaction(s) of the phosphorylated regulator at the cognate binding sites. The fact that these sites within the regulatory region of respective genes do not bind in vitro with either unphosphorylated or phosphorylation-deficient PhoP protein is consistent with phosphorylation-dependent assembly of the transcription initiation complex leading to in vivo transcriptional activation. Together, these results reveal so far unknown molecular mechanisms of how PhoP contributes to M. tuberculosis cell wall composition by regulating complex lipid biosynthesis.

Development of a novel quantitative real-time assay using duplex mutation primers for rapid detection of Candida species

We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species.

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer–template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.

Mono-ubiquitination Drives Nuclear Export of the Human DCN1-like Protein hDCNL1

Conjugation of Nedd8 to a cullin protein, termed neddylation, is an evolutionarily conserved process that functions to activate the cullin-RING family E3 ubiquitin ligases, leading to increased proteasomal degradation of a wide range of substrate proteins. Recent emerging evidence demonstrates that cellular neddylation requires the action of Dcn1, which, in humans, consists of five homologues designated as hDCNL1-5. Here we revealed a previously unknown mechanism that regulates hDCNL1. In cultured mammalian cells ectopically expressed hDCNL1 was mono-ubiquitinated predominantly at K143, K149, and K171. Using a classical chromatographic purification strategy, we identified Nedd4-1 as an E3 ligase that can catalyze mono-ubiquitination of hDCNL1 in a reconstituted ubiquitination system. In addition, the hDCNL1 N-terminal ubiquitin-binding domain is necessary and sufficient to mediate mono-ubiquitination. Finally, fluorescence microscopic and subcellular fractionation analyses revealed a role for mono-ubiquitination in driving nuclear export of hDCNL1. Taken together, these results suggest a mono-ubiquitination-mediated mechanism that governs nuclear-cytoplasmic trafficking of hDCNL1, thereby regulating hDCNL1-dependent activation of the cullin-RING E3 ubiquitin ligases in selected cellular compartments.

Development of a novel quantitative real-time assay using self-reporting duplex mutation primers for detection of HCV

Objective To establish a novel real-time PCR method to detect HCV RNA using Selfreporting duplex mutation primers.Methods The recombinant vector pMD18-T-HCV 5′-NCR was used as the calibrator.The Self-reporting duplex mutation primers were designed according to the gene sequence.And then the PCR reaction system was optimized and evaluated.The specificity,sensitivity and reproducibility of real-time PCR were estimated,The serum specimens from 90 cases(30 cases of HCV,30 cases of other viral hepatitis and 30 healthy volunteers) were tested with this real-time PCR; Results were compared with those obtained using a commercial TaqMan kit.Results The assay was established.It showed linearity over a wide range from 20 - 109 IU/ml.Intra-experimental coefficients of variation(CVs) were 1.37% -4.59%,and inter-experimental CVs were 1.58% -4.81%,respectively.There was no significant difference of HCV genome number tested by the two methods(R2 = 0.95) in 30 hepatitis C patients; HCV DNA was not detected in any serum samples of 30 healthy volunteers by the two methods.The specificity was 100%(60/60).All the samples in patients with clinically confirmed HCV infections showed HCV RNA positive.There wass good correlation between the quantitaive results and results obtained using the commercial TaqMan kit.Conclusions It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for HCV.The establishment of this assay provided alternative technology for clinical diagnosis or therapeutic drug monitoring in the field of HCV infection and epidemiologic survey. Key words: Hepacivirus; Polymerase chain reaction; RNA,viral

Electrostatic Interactions Involving the Extreme C Terminus of Nuclear Export Factor CRM1 Modulate Its Affinity for Cargo

The toroid-shaped nuclear protein export factor CRM1 is constructed from 21 tandem HEAT repeats, each of which contains an inner (B) and outer (A) α-helix joined by loops. Proteins targeted for export have a nuclear export signal (NES) that binds between the A-helices of HEAT repeats 11 and 12 on the outer surface of CRM1. RanGTP binding increases the affinity of CRM1 for NESs. In the absence of RanGTP, the CRM1 C-terminal helix, together with the HEAT repeat 9 loop, modulates its affinity for NESs. Here we show that there is an electrostatic interaction between acidic residues at the extreme distal tip of the C-terminal helix and basic residues on the HEAT repeat 12 B-helix that lies on the inner surface of CRM1 beneath the NES binding site. Small angle x-ray scattering indicates that the increased affinity for NESs generated by mutations in the C-terminal helix is not associated with large scale changes in CRM1 conformation, consistent with the modulation of NES affinity being mediated by a local change in CRM1 near the NES binding site. These data also suggest that in the absence of RanGTP, the C-terminal helix lies across the CRM1 toroid in a position similar to that seen in the CRM1-Snurportin crystal structure. By creating local changes that stabilize the NES binding site in its closed conformation and thereby reducing the affinity of CRM1 for NESs, the C-terminal helix and HEAT 9 loop facilitate release of NES-containing cargo in the cytoplasm and also inhibit their return to the nucleus.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

Structural and Functional Relationships between the Lectin and Arm Domains of Calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys(32)) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.