scholarly journals Binding of Pyridine Nucleotide Coenzymes to the β-Subunit of the Voltage-sensitive K+Channel

2001 ◽  
Vol 276 (15) ◽  
pp. 11812-11820 ◽  
Author(s):  
Si-Qi Liu ◽  
Hongjun Jin ◽  
Albert Zacarias ◽  
Sanjay Srivastava ◽  
Aruni Bhatnagar
Keyword(s):  
Pyridine Nucleotide ◽  
K Channel ◽  
Β Subunit ◽  
Wild Type ◽  
Ionic Strength Dependence ◽  
Coenzyme Binding ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tetrameric Structure

The β-subunit of the voltage-sensitive K+(Kv) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of Kvβ2 shows NADP bound in its active site. Here we report that Kvβ2 displays a high affinity for NADPH (Kd= 0.1 μm) and NADP+(Kd= 0.3 μm), as determined by fluorometric titrations of the recombinant protein. The Kvβ2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, theKdNADPHof Q214R was 10-fold greater than the wild-type protein. TheKdNADPHwas unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease inKdNADPH, shifted the pKaofKdNADPHfrom 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of Kvβ2-nucleotide complexes may be formedin vivo.

scholarly journals Identification and Analysis of a Hyperactive Mutant Form of Drosophila P-Element Transposase

Genetics ◽  
2002 ◽  
Vol 162 (1) ◽  
pp. 217-227 ◽  
Author(s):  
Eileen L Beall ◽  
Matthew B Mahoney ◽  
Donald C Rio
Keyword(s):  
Dna Damage ◽  
Dna Cleavage ◽  
P Element ◽  
In Vitro Assays ◽  
Mutant Form ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Abstract Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI3-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

Generation of Novel, High Titre, Multi-Cistronic Retroviral Vectors Which Simultaneously Express Individual Proteins in Different Subcellular Locations of Hematopoietic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5255-5255
Author(s):  
Michael D. Milsom ◽  
Catherine Gavin ◽  
Rebecca Baldwin ◽  
Geoff Margison ◽  
Leslie J. Fairbairn
Keyword(s):  
Gene Therapy ◽  
Dna Methyltransferase ◽  
Growth Factor Receptor ◽  
Retroviral Vectors ◽  
High Titre ◽  
Target Cells ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Abstract As gene therapy strategies become ever more sophisticated, there is a need for vectors which efficiently deliver and express more than one gene product to target cells e.g. to enable in vivo selection; to correct multiple genetic loci; to provide a suicide mechanism for safety purposes. Employing alternate codon usage, we have synthesised a novel version of the FMDV 2A self-processing moiety which encodes the same primary amino-acid sequence as the wild type protein (and which possesses equivalent cleavage activity to the wild type protein). By including this synthetic FMDV 2A sequence in retroviral vectors which also contain the wild type sequence, we are able to facilitate the efficient co-translational separation of 3 different proteins encoded by a single transcript. This vector configuration also permits the inclusion of an IRES sequence and thus expression of a fourth protein. Using this technology we have created two tetracistronic retroviral vectors which express eGFP; O6-methylguanine DNA-methyltransferase and truncated human nerve growth factor receptor courtesy of 2A mediated cleavage, and a mitochondrially targeted dsRED via an IRES. Stable amphotropic producer lines containing these vectors generated supernatant which contained a high titre of retroviral particles (>106 IU/ml) which was sufficient for the transduction of K562 human erythroleukemic cells. Analysis of transduced cells confirmed that all four proteins were expressed individually and segragated to their correct subcellular locations. Furthermore, limiting dilution assay revealed that proteins expressed courtesy of the 2A sequences were still generated at high levels, even when cells were infected with a low MOI of virus. We propose that this technology will allow the creation of more sophisticated vectors for use in gene therapy of hematopoietic and other cells.

Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA

2008 ◽  
Vol 414 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Allison Lewin ◽  
Allister Crow ◽  
Christopher T. C. Hodson ◽  
Lars Hederstedt ◽  
Nick E. Le Brun
Keyword(s):  
Active Site ◽  
Protein Unfolding ◽  
Cysteine Residues ◽  
Wild Type ◽  
Pka Values ◽  
Active Site Cysteine ◽  
Reduction Potentials ◽  
Type Protein ◽  
Wild Type Protein

The thiol–disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pKa values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in that they are both high (>8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid–base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pKa values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, ΔpKa values of −2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited an increase of +40 mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to the oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.

scholarly journals Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0128954 ◽  
Author(s):  
Saara Laulumaa ◽  
Tuomo Nieminen ◽  
Mari Lehtimäki ◽  
Shweta Aggarwal ◽  
Mikael Simons ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Neutron Scattering ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Peripheral Membrane Protein

Two novel mutations in Gli-similar 3 in patients with congenital hypothyroidism and thyroid dysgenesis

2021 ◽  
Author(s):  
Jie Lan ◽  
Chunhui Sun ◽  
Xinping Liang ◽  
Ruixin Ma ◽  
Yuhua Ji ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Transcriptional Activation ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Thyroid Dysgenesis ◽  
Type Protein ◽  
Wild Type Protein

Abstract Background: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). As variants of the transcription factor Gli-similar 3 (GLIS3) have been associated with CH and GLIS3 is one of candidate genes of TD, we screened and characterized GLIS3 mutations in Chinese patients with CH and TD.Methods: To detect mutations, we sequenced all GLIS3 exons in the peripheral blood genomic DNA isolated from 50 patients with TD and 100 healthy individuals. Wild-type and mutant expression vectors of Glis3 were constructed. Quantitative real-time PCR, western blotting, and double luciferase assay were performed to investigation the effect of the mutations on GLIS3 protein function and transcriptional activation.Results: Two novel heterozygous missense mutations, c.2710G>A (p.G904R) and c.2507C>A (p.P836Q), were detected in two unrelated patients. Functional studies revealed that p.G904R expression was 59.95% lower and p.P836Q was 31.23% lower than wild-type GLIS3 mRNA expression. The p.G904R mutation also resulted in lower GLIS3 protein expression compared with that encoded by wild-type GLIS3. Additionally, the luciferase reporter assay revealed that p.G904R mediated impaired transcriptional activation compared with the wild-type protein (p < 0.05) but did not have a dominant-negative effect on the wild-type protein.Conclusions: We for the first time screened and characterized the function of GLIS3 mutations in Chinese individuals with CH and TD. Our study not only broadens the GLIS3 mutation spectrum, but also provides further evidence that GLIS3 defects cause TD.

scholarly journals Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-Crystallin on Chaperone Function and Anti-Apoptotic Activity

2021 ◽  
Vol 22 (19) ◽  
pp. 10771
Author(s):  
Sundararajan Mahalingam ◽  
Srabani Karmakar ◽  
Puttur Santhoshkumar ◽  
Krishna K. Sharma
Keyword(s):  
Deletion Mutant ◽  
Mutant Protein ◽  
Wild Type ◽  
Chaperone Function ◽  
Type Protein ◽  
Wild Type Protein ◽  
Cytotoxicity Studies ◽  
Αb Crystallin ◽  
Apoptotic Activity ◽  
Subunit Organization

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

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