Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

2013 ◽  
Vol 1834 (12) ◽  
pp. 2782-2794 ◽  
Author(s):  
Eiji Ohmae ◽  
Yurina Miyashita ◽  
Shin-ichi Tate ◽  
Kunihiko Gekko ◽  
Soichiro Kitazawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Active Site ◽  
Wild Type ◽  
Enzymatic Function ◽  
Type Protein ◽  
Wild Type Protein

scholarly journals Identification of an autoinhibitory region in the activation loop of the Mos protein kinase.

1996 ◽  
Vol 16 (7) ◽  
pp. 3472-3479 ◽  
Author(s):  
S C Robertson ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Active Site ◽  
Kinase Activity ◽  
Molecular Model ◽  
Wild Type ◽  
Three Dimensional Model ◽  
Loop Region ◽  
Type Protein ◽  
Wild Type Protein ◽  
Ability To Act

The Mos protein is a serine/threonine protein kinase which acts to regulate progression through meiosis in vertebrate oocytes. Although Mos function is dependent on its ability to act as a protein kinase, little is known about the factors which regulate Mos kinase activity. To understand the mechanism by which Mos kinase activity is regulated, we have used molecular modeling to construct a three-dimensional model of Mos based on the crystallographic coordinates of cyclic AMP-dependent kinase (PKA). This model identified a loop in Mos which is positioned near the active site and appears capable of blocking substrate access to the active site. Mutagenesis was used to construct altered forms of the Mos protein with deletions of parts or all of the loop. In vitro kinase assays showed that Mos proteins with the loop removed had up to a fourfold increase in kinase activity compared with the wild-type protein, indicating that the loop acts in an autoinhibitory manner for Mos kinase activity. Point mutations were also made on individual residues of the loop which were determined from the molecular model to be capable of reaching the active site. Determination of the kinase activities of these mutants showed that individual mutations in the loop region are capable of either increasing or decreasing kinase activity with regard to the wild-type protein. These data suggest that the loop identified in Mos acts as an autoinhibitor of kinase activity.

scholarly journals Cloning of T4 gene 32 and expression of the wild-type protein under lambda promoter PL regulation in Escherichia coli.

1986 ◽  
Vol 83 (23) ◽  
pp. 8844-8848 ◽  
Author(s):  
Y. Shamoo ◽  
H. Adari ◽  
W. H. Konigsberg ◽  
K. R. Williams ◽  
J. W. Chase
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Gene 32

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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