Thermal stability of wild type and disulfide bridge containing mutant of poplar plastocyanin

2004 ◽  
Vol 112 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Rita Guzzi ◽  
Laura Andolfi ◽  
Salvatore Cannistraro ◽  
Martin Ph. Verbeet ◽  
Gerard W. Canters ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Disulfide Bridge ◽  
Wild Type ◽  
Thermal Stability Of

scholarly journals LAC4 IS THE STRUCTURAL GENE FOR β-GALACTOSIDASE IN KLUYVEROMYCES LACTIS

Genetics ◽  
1981 ◽  
Vol 98 (4) ◽  
pp. 729-745
Author(s):  
R Michael Sheetz ◽  
Robert C Dickson
Keyword(s):  
Thermal Stability ◽  
Structural Gene ◽  
Gel Electrophoresis ◽  
Kluyveromyces Lactis ◽  
Two Dimensional ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Nonsense Suppressor ◽  
Hydrolysis Of ◽  
Thermal Stability Of

ABSTRACT Using genetic and biochemical techniques, we have determined that β-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of β-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac+ revertant, bearing lac4–14 and this unlinked suppressor, has subnormal levels of β-galactosidase activity, and the Km for hydrolysis of o-nitrophenyl-β, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of β-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acryl-amide gel electrophoresis shows that strains bearing lac4–23 and lac4–30 contain an inactive β-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is obs0ervable in lac4–8, lac4–14 or lac4–29 mutants; (6) of all lac4 mutants, only those bearing lac4–23 or lac4–30 contain a protein that cross-reacts with anti-β-galactosidase antibody, a finding consistent with the previous result; and (7) β-galactosidase activity in several Lac+ revertants of strains carrying lac4–23 or lac4–30 has greatly decreased thermostability.

scholarly journals Rational Protein Engineering to Increase the Activity and Stability of IsPETase Using the PROSS Algorithm

Polymers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 3884
Author(s):  
Andrew Rennison ◽  
Jakob R. Winther ◽  
Cristiano Varrone
Keyword(s):  
Thermal Stability ◽  
Enzyme Stability ◽  
Fold Increase ◽  
Wild Type ◽  
Bioinformatic Tools ◽  
Mild Conditions ◽  
Degradation Activity ◽  
Packaging Industry ◽  
The Stability ◽  
Thermal Stability Of

Polyethylene terephthalate (PET) is the most widely used polyester plastic, with applications in the textile and packaging industry. Currently, re-moulding is the main path for PET recycling, but this eventually leads to an unsustainable loss of quality; thus, other means of recycling are required. Enzymatic hydrolysis offers the possibility of monomer formation under mild conditions and opens up alternative and infinite recycling paths. Here, IsPETase, derived from the bacterium Ideonella sakaiensis, is considered to be the most active enzyme for PET degradation under mild conditions, and although several studies have demonstrated improvements to both the stability and activity of this enzyme, stability at even moderate temperatures is still an issue. In the present study, we have used sequence and structure-based bioinformatic tools to identify mutations to increase the thermal stability of the enzyme so as to increase PET degradation activity during extended hydrolysis reactions. We found that amino acid substitution S136E showed significant increases to activity and stability. S136E is a previously unreported variant that led to a 3.3-fold increase in activity relative to wild type.

scholarly journals Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme

2005 ◽  
Vol 386 (3) ◽  
pp. 599-605 ◽  
Author(s):  
Ricardo FRANCO ◽  
Guangyue BAI ◽  
Vesna PROSINECKI ◽  
Filipa ABRUNHOSA ◽  
Gloria C. FERREIRA ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protoporphyrin Ix ◽  
Titration Calorimetry ◽  
Wild Type ◽  
Scanning Calorimetry ◽  
Binding Reaction ◽  
Key Factor ◽  
Single Transition ◽  
Thermal Stability Of ◽  
Denaturation Process

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the chelation of Fe(II) into the protoporphyrin IX ring. The energetics of the binding between murine ferrochelatase and mesoporphyrin were determined using isothermal titration calorimetry, which revealed a stoichiometry of one molecule of mesoporphyrin bound per protein monomer. The binding is strongly exothermic, with a large intrinsic enthalpy (ΔH=−97.1 kJ · mol−1), and is associated with the uptake of two protons from the buffer. This proton transfer suggests that hydrogen bonding between ferrochelatase and mesoporphyrin is a key factor in the thermodynamics of the binding reaction. Differential scanning calorimetry thermograms indicated a co-operative two-state denaturation process with a single transition temperature of 56 °C for wild-type murine ferrochelatase. An increase in the thermal stability of ferrochelatase is dependent upon mesoporphyrin binding. Similarly, murine ferrochelatase variants, in which the active site Glu-289 was replaced by either glutamine or alanine and, when purified, contained specifically-bound protoporphyrin, exhibited enhanced protein stability when compared with wild-type ferrochelatase. However, in contrast with the wild-type enzyme, the thermal denaturation of ferrochelatase variants was best described as a non-co-operative denaturation process.

The CPVT-associated RyR2 mutation G230C enhances store overloadinduced Ca2+ release and destabilizes the N-terminal domains

2013 ◽  
Vol 454 (1) ◽  
pp. 123-131 ◽  
Author(s):  
Yingjie Liu ◽  
Lynn Kimlicka ◽  
Florian Hiess ◽  
Xixi Tian ◽  
Ruiwu Wang ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Lipid Bilayers ◽  
Polymorphic Ventricular Tachycardia ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Disease Mechanism ◽  
Structural Impact ◽  
Life Threatening ◽  
Thermal Stability Of ◽  
Terminal Domains

CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed after-depolarizations) that are induced by spontaneous Ca2+ release during SR (sarcoplasmic reticulum) Ca2+ overload, a process also known as SOICR (store-overload-induced Ca2+ release). A number of mutations in the cardiac ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2 mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to luminal or luminal/cytosolic Ca2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca2+ sensitivity of single RyR2 channels in lipid bilayers. This observation led to the suggestion of a SOICR-independent disease mechanism for the G230C mutation. However, the cellular impact of this mutation on SOICR is yet to be determined. To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell Ca2+ imaging, we found that the G230C mutation markedly enhanced the propensity for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation increased the sensitivity of single RyR2 channels to both luminal and cytosolic Ca2+ activation and the Ca2+-dependent activation of [3H]ryanodine binding. In addition, the G230C mutation decreased the thermal stability of the N-terminal region (amino acids 1–547) of RyR2. These data suggest that the G230C mutation enhances the propensity for SOICR by sensitizing the channel to luminal and cytosolic Ca2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.

The Ordered Phase Formation in Ti-Al-Ga Alloys

1970 ◽  
Vol 28 ◽  
pp. 440-441
Author(s):  
Shiro Fujishiro ◽  
Harold L. Gegel
Keyword(s):  
Thermal Stability ◽  
High Temperature ◽  
Titanium Alloys ◽  
Growth Kinetics ◽  
Stoichiometric Composition ◽  
Good Potential ◽  
Alpha Titanium ◽  
Cold Rolled ◽  
Kinetics Of ◽  
Thermal Stability Of

Ordered-alpha titanium alloys having a DO19 type structure have good potential for high temperature (600°C) applications, due to the thermal stability of the ordered phase and the inherent resistance to recrystallization of these alloys. Five different Ti-Al-Ga alloys consisting of equal atomic percents of aluminum and gallium solute additions up to the stoichiometric composition, Ti3(Al, Ga), were used to study the growth kinetics of the ordered phase and the nature of its interface.The alloys were homogenized in the beta region in a vacuum of about 5×10-7 torr, furnace cooled; reheated in air to 50°C below the alpha transus for hot working. The alloys were subsequently acid cleaned, annealed in vacuo, and cold rolled to about. 050 inch prior to additional homogenization

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