Thermodynamic effects of reduction of the active-site disulfide of Escherichia coli thioredoxin explored by differential scanning calorimetry

Biochemistry ◽  
1994 ◽  
Vol 33 (12) ◽  
pp. 3688-3692 ◽  
Author(s):  
John E. Ladbury ◽  
Nand Kishore ◽  
Homme W. Hellinga ◽  
Richard Wynn ◽  
Julian M. Sturtevant
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Active Site ◽  
Scanning Calorimetry ◽  
Thermodynamic Effects

Thermotoga neapolitana Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in Escherichia coli

1998 ◽  
Vol 64 (7) ◽  
pp. 2357-2360 ◽  
Author(s):  
J. Michael Hess ◽  
Vladimir Tchernajenko ◽  
Claire Vieille ◽  
J. Gregory Zeikus ◽  
Robert M. Kelly
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Gel Filtration ◽  
Xylose Isomerase ◽  
Thermotoga Neapolitana ◽  
Thermal Transitions ◽  
Scanning Calorimetry ◽  
Catalytically Active ◽  
The Stability ◽  
Tetrameric Form

ABSTRACT The xylA gene from Thermotoga neapolitana5068 was expressed in Escherichia coli. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.

scholarly journals Growth measurement of Escherichia coli by differential scanning calorimetry

2014 ◽  
Vol 8 (16) ◽  
pp. 1656-1662 ◽  
Author(s):  
Rangel-Vargas Esmeralda ◽  
A. Gmez-Aldapa Carlos ◽  
R. Villagmez-Ibarra Jos ◽  
M. Santos-Lpez Eva ◽  
Rodriguez-Miranda Jesus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Scanning Calorimetry ◽  
Growth Measurement

scholarly journals Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases

2003 ◽  
Vol 374 (3) ◽  
pp. 785-792 ◽  
Author(s):  
Irene KARANTZENI ◽  
Carmen RUIZ ◽  
Chin-Chi LIU ◽  
Vince J. LiCATA
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Thermal Denaturation ◽  
Dna Polymerases ◽  
Large Fragment ◽  
Scanning Calorimetry ◽  
Thermus Aquaticus ◽  
Taq Polymerase ◽  
Nuclease Domain

Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5′ nuclease domain. Removal of the 5′ nuclease domains produces the ‘large fragment’ domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature Tm of 37 and 100 °C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5′ nuclease domains of each melting separately. The 5′ nuclease domain of Taq denatures as a separate peak, 10 °C before the Klentaq domain. Melting of the 5′ nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5′ nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower Tm values, but almost identical van't Hoff enthalpy ΔH values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.

Differential scanning calorimetry of the irreversible denaturation of Escherichia coli glucosamine-6-phosphate deaminase

Biochemistry ◽  
1993 ◽  
Vol 32 (14) ◽  
pp. 3644-3648 ◽  
Author(s):  
Andres Hernandez-Arana ◽  
Arturo Rojo-Dominguez ◽  
Myriam M. Altamirano ◽  
Mario L. Calcagno
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Scanning Calorimetry ◽  
Irreversible Denaturation
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