Elucidating the role of membranes in modulating the thermodynamics and kinetics of substrate binding to Thromboxane Synthase

Snehita Sri Varma, -

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https://hdl.handle.net/2142/44805 Copy

Description

Title

Elucidating the role of membranes in modulating the thermodynamics and kinetics of substrate binding to Thromboxane Synthase

Author(s)

Snehita Sri Varma, -

Issue Date

2013-05-28T19:20:36Z

Director of Research (if dissertation) or Advisor (if thesis)

Das, Aditi

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• Thromboxane synthase
• redox potential
• cyanide binding
• coupled activity assay

Abstract

Thromboxane synthase (CYP5A1) is a membrane-bound cytochrome P450. It synthesizes thromboxane A2 (TXA2) from PGH2, which is a potent mediator of platelet aggregation and vasoconstriction. TXA2 has been implicated in a wide range of pathophysiological disease conditions. Though it is a membrane protein, most of its biochemical characterization has been performed in soluble reconstituted systems. Membranes have been shown to play an important role in the structure and function of membrane proteins. This work presents the first report on membrane incorporated CYP5A1 and its biochemical characterization. To achieve this, native-like lipid bilayers called nanodiscs were used to obtain monomeric and stabilized protein. The protein was successfully expressed in a bacterial expression system and the purified protein was incorporated into nanodiscs. This system was functionally characterized using CO binding, substrate binding and activity assays. The specific activity of the protein was evaluated both in nanodiscs and detergent solubilized system and was shown to increase by 100% in Nanodiscs. In order to understand the role of the membrane in modulating the electrostatic environment of the protein, redox potentials of substrate free and substrate bound CYP5A1 in membranes was measured. This is also the first account of an endoperoxide substrate like PGH2 bound to a protein. We noticed that the redox potential of the protein in Nanodiscs were not very different from those in detergents showing lack of modulation of the active site electrostatics by the membrane environment. The kinetics of substrate analog (U44069 and U46619) and cyanide binding to CYP5A1-nanodiscs was measured using stopped flow to evaluate the conformational change in the protein in lipid bilayers of Nanodiscs. It was determined that the kinetic parameters of binding of the substrate analogs to CYP5A1-nanodisc system were different from detergent solubilized state. Moreover, the accessibility of cyanide to the active site of the protein also changed in CYP5A1-nanodiscs as compared to detergent solubilized systems. These results show that the conformation of CYP5A1 is different in Nanodiscs which may account for the increased activity of the protein in the lipid bilayers.

Graduation Semester

2013-05

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http://hdl.handle.net/2142/44805

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Copyright 2013 - Snehita Sri Varma

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