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Thermodynamics of Complexation: Variant R15L hGST A1-1 binding to ANS and GSO3Dobreva, Marina Alexandrova 31 October 2006 (has links)
Student Number : 0009716H -
MSc dissertation -
School of Molecular and Cell Biology -
Faculty of Science / Positive charges play an important role in the active site of a variety of proteins including
alpha class GST. The presence of Arg15 at the subunit interface between the H- and Gsite
of hGST A1-1 was shown to be important for the catalytic function of the enzyme,
thus providing an electrostatic potential in the G-site. This electrostatic potential favours
ionisation and activation of GSH bound to hGST A1-1. Although much is known
regarding the catalytic function of GSTs, little is known of the effect of Arg15 on the
binding of glutathione conjugates and nonsubstrate ligands. Others (Matulis and Lovrien,
1998) have pointed out that binding of the nonsubstrate ligand ANS is accomplished via
ion pair formation with a charged residue such as Arg. Therefore, in order to evaluate the
importance of the positive charge provided by the side chain of Arg15 in the active site
of hGST A1-1 protein engineering techniques were employed to generate an R15L
variant. The variant does not display a detectable difference in the secondary or tertiary
structural content relative to the wild type hGST A1-1 protein. The catalytic activity of
the R15L variant is, however, substantially reduced indicating local tertiary structural
changes at and possibly near the active site. In the presence of ANS the fluorescence
spectra of the variant R15L protein had a significantly lower intensity relative to wild
type protein indicating either increased exposure of hydrophobic surface area at the Hsite
and/or reduced number of ANS molecules bound (i.e., reduced affinity). Although
ANS is displaced by GSO3
- in the wild type protein, no such displacement is observed for
the R15L variant protein. Isothermal titration calorimetry experiments for GSO3
- and
ANS binding to R15L indicated that the variant protein binds GSO3
- more tightly (higher
affinity) than ANS (lower affinity). The above information, taken together, argues in
favour of both molecules (ANS and GSO3
-) occupying the active site of R15L
simultaneously. Despite the fact that there is no experimental crystal structure of hGST
A1-1 complexed with either of the ligands, the placement of ANS was argued (Dirr et al.,
2005) to be at the H-site and of GSO3
- at the G-site of the protein. The binding of ANS is
both enthalpically and entropically favourable over the temperature range investigated (5
to 25°C) indicating expulsion of water molecules from the active site as well as the lack
of a localised C-terminal #1;9 helix. The hydrophobic character of both the ligand and the site which accommodates it (H-site) contribute favourably towards the reaction enthalpy.
ANS is conformationaly more constrained than GSO3
- when found free in solution, thus
exhibiting more favourable conformational entropy change upon binding to R15L variant.
The binding of GSO3
- is enthalpically favourable but entropically opposed. The expulsion
of water molecules from the G-site, which accommodates GSO3
-, is insufficient to
compensate for the loss of conformational entropy of both the ligand and C-terminal #1;9
helix, which becomes localised upon binding. However, favourable enthalpic
contributions arise from the formation of hydrogen bonds, salt links and van der Waals
contacts between the R15L variant and the ligand and the C-terminal #1;9 helix. Arg15
forms a salt link with Glu104 of the neighbouring domain and therefore provided an ideal
opportunity to evaluate the role of Arg15 and its impact on interdomain stability.
Conformational stability and unfolding studies of the R15L variant, monitored via
fluorescence, indicated that the variant deviates from the model of a two-state transition.
There are no stable thermodynamic intermediate(s) observed, but the increase in the mvalue
is an indicator of a transition state which is more stable against urea relative to the
wild type protein. Kinetically (Wallace et al., 1998b), the wild type hGST A1-1 follows
a three-state unfolding transition where the kinetic intermediate is defined as having
undergone conformational changes at the domain-domain interface and the C-terminal
region. The substitution of Arg15 with Leu may have impacted on the packing of the
native form of the variant R15L relative to the wild type GST A1-1, thus rendering the
R15L protein more stable against urea. Arg15, therefore, may have an important role at
the interdomain interface of GSTs.
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La guerre de Cent ans et le prince chevalier le "bon duc" Louis II de Bourbon : 1337-1410.Troubat, Olivier. January 2001 (has links)
Texte remanié de: Th. doct.--Hist.--Lyon 2, 1991. / Bibliogr. p. 25-87. L'ouvrage porte par erreur : ISSN 0395-1596.
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Die Grafschaft Hohenlohe im Dreißigjährigen Krieg : eine erfahrungsgeschichtliche Untersuchung zu Herrschaft und Untertanen /Kleinehagenbrock, Frank. January 2003 (has links)
Texte remanié de: Diss.--Fakultät für Philosophie und Geschichte--Tübingen--Eberhard-Karls-Universität, 2002. / Bibliogr. p. XV-LIV. Index.
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"Die Profiantirung der keyserlichen Armaden ahnbelangendt" : Studien zur Versorgung der kaiserlichen Armee 1634-35 /Polh, Jürgen, January 1900 (has links)
Diss.--Kiel--Christian-Albrechts Universität, 1991. / Bibliogr. p. 210-218.
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Treue Deserteure : das kursächsische Militär und der Siebenjährige Krieg /Salisch, Marcus von. January 2009 (has links)
Dissertation--München--Universität der Bundeswehr, 2007. / Bibliogr. p. 309-329.
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ANSDA - An analytic assesment of its processesLundström, Fredrik, Rondin, Joakim January 2013 (has links)
This is the final report of ANSDA – An analytic assessment of its processes written as a bachelor thesis in the fall of 2013 by students at Linköpings Universitet. It is an analysis of the incident evaluation process used by LFV: ANS-DA. The thesis aimed to find areas where the process could be optimized in regards to time-consumption and efficency in dealing with errors in Air Traffic Control procedure, which are observed when an anomaly in the system occurs.
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Die Kosten des Westfälischen Friedenskongresses : eine strukturgeschichtliche Untersuchung /Bosbach, Franz. January 1984 (has links)
Diss. : Philosophische Fakultät : Bonn : 1981. - Bibliogr. p. 234-244. Index. -
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Okkupation und Regionalismus : die preussischen Westprovinzen im Siebenjährigen Krieg /Carl, Horst, January 1993 (has links)
Texte remanié de: Diss.--Geschichtswissenschaftliche Fakultät--Tübingen--Eberhard-Karls-Universität, 1990.
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Die Kipper und Wipper als publizistisches Ereignis (1620-1626) : eine Studie zu den Strukturen öffentlicher Kommunikation im Zeitalter des Dreissigjährigen Krieges /Rosseaux, Ulrich. January 1900 (has links)
Diss.--Philosophische Fakultät--Bonn--Rheinische Friedrich-Wilhelms-Universität, Wintersemester 1999/2000. / Bibliogr. p. 540-588. Index.
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Die Peripetie des Siebenjährigen Krieges : der Herbstfeldzug 1760 in Sachsen und der Winterfeldzug 1760/61 in Hessen /Lindner, Thomas, January 1900 (has links)
Diss.--Philosophische Fakultät--Universität Köln, 1993. / Bibliogr. p. 243-255.
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