scholarly journals pH-Induced Local Unfolding of the Phl p 6 Pollen Allergen From cpH-MD

2021 ◽  
Vol 7 ◽  
Author(s):  
Florian Hofer ◽  
Anna S. Kamenik ◽  
Monica L. Fernández-Quintero ◽  
Johannes Kraml ◽  
Klaus R. Liedl
Keyword(s):  
Allergic Sensitization ◽  
Proteolytic Cleavage ◽  
Pollen Allergen ◽  
Current View ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Endosomal Acidification ◽  
Local Unfolding ◽  
The Impact

Susceptibility to endosomal degradation is a decisive contribution to a protein's immunogenicity. It is assumed that the processing kinetics of structured proteins are inherently linked to their probability of local unfolding. In this study, we quantify the impact of endosomal acidification on the conformational stability of the major timothy grass pollen allergen Phl p 6. We use state of the art sampling approaches in combination with constant pH MD techniques to profile pH-dependent local unfolding events in atomistic detail. Integrating our findings into the current view on type 1 allergic sensitization, we characterize local protein dynamics in the context of proteolytic degradation at neutral and acidic pH for the wild type protein and point mutants with varying proteolytic stability. We analyze extensive simulation data using Markov state models and retrieve highly reliable thermodynamic and kinetic information at varying pH levels. Thereby we capture the impact of endolysosomal acidification on the structure and dynamics of the Phl p 6 mutants. We find that upon protonation at lower pH values, the conformational flexibilities in key areas of the wild type protein, i.e., T-cell epitopes and early proteolytic cleavage sites, increase significantly. A decrease of the pH even leads to local unfolding in otherwise stable secondary structure elements, which is a prerequisite for proteolytic cleavage. This effect is even more pronounced in the destabilized mutant, while no unfolding was observed for the stabilized mutant. In summary, we report detailed structural models which rationalize the experimentally observed cleavage pattern during endosomal acidification.

Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop

2002 ◽  
Vol 363 (2) ◽  
pp. 403-409 ◽  
Author(s):  
Yasuhiro ARII ◽  
Masaaki HIROSE
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Proteolytic Cleavage ◽  
Insertion Reaction ◽  
Wild Type ◽  
Order Rate ◽  
Transition Mechanism ◽  
Type Protein ◽  
Wild Type Protein ◽  
Insertion Mechanism

A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1—P1′ cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Mol. Biol. 315, 113–120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented futher cleavage of the P1—P1′ pre-cleaved R339T mutant by subtilisin, which cleaved the second P8—P7 loop site in the P1—P1′ pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1—P1′ and double P1—P1′/P8—P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8—P7 site in the immediate intermediate after the P1—P1′ cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0×10−3/s in the R339T mutant. The second-order rate constant for the P8—P7 cleavage in the immediate P1—P1′ cleavage product for the R339T mutant was >10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.

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.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr103 affects rate and product distribution

2004 ◽  
Vol 381 (2) ◽  
pp. 365-372 ◽  
Author(s):  
Benjamin S. RAYNER ◽  
Roland STOCKER ◽  
Peter A. LAY ◽  
Paul K. WITTING
Keyword(s):  
Linoleic Acid ◽  
Lipid Oxidation ◽  
Octadecadienoic Acid ◽  
Total Yield ◽  
Product Distribution ◽  
Chemical Oxidation ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Lipid Oxidation Products

Mb (myoglobin) plus H2O2 catalyses the oxidation of various substrates via a peroxidase-like activity. A Y103F (Tyr103→Phe) variant of human Mb has been constructed to assess the effect of exchanging an electron-rich oxidizable amino acid on the peroxidase activity of human Mb. Steady-state analyses of reaction mixtures containing Y103F Mb, purified linoleic acid and H2O2 revealed a lower total yield of lipid oxidation products than mixtures containing the wild-type protein, consistent with the reported decrease in the rate constant for reaction of Y103F Mb with H2O2 [Witting, Mauk and Lay (2002) Biochemistry 41, 11495–11503]. Irrespective of the Mb employed, lipid oxidation yielded 9(R/S)-HODE [9(R,S)-hydroxy-10E,12Z-octadecadienoic acid] in preference to 13(R/S)-HODE [13(R,S)-hydroxy-9Z,11E-octadecadienoic acid], while 9- and 13-keto-octadecadienoic acid were formed in trace amounts. However, lipid oxidation by the Y103F variant of Mb proceeded with a lower Vmax value and an increased Km value relative to the wild-type control. Consistent with the increased Km, the product distribution from reactions with Y103F Mb showed decreased selectivity compared with the wild-type protein, as judged by the decreased yield of 9(S)-relative to 9(R)-HODE. Together, these data verify that Tyr103 plays a significant role in substrate binding and orientation in the haem pocket of human Mb. Also, the midpoint potential for the Fe(III)/(II) one-electron reduction was shifted slightly, but significantly, to a higher potential, confirming the importance of Tyr103 to the hydrogen-bonding network involving residues that line the haem crevice of human Mb.

scholarly journals S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) ReduceIn VitroInhibition by Fluconazole

2012 ◽  
Vol 56 (4) ◽  
pp. 2099-2107 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Jonathan G. L. Mullins ◽  
Claire M. Hull ◽  
Josie E. Parker ◽  
David C. Lamb ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Point Mutations ◽  
Triazole Ring ◽  
Wild Type ◽  
Protein Activity ◽  
Content Type ◽  
Mutant Proteins ◽  
Type Protein ◽  
Wild Type Protein ◽  
Binding Spectra

ABSTRACTThe effects of S279F and S279Y point mutations inCandida albicansCYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (Ks, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (Ks, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higherKdvalues observed.

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