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The biophysical characterization of ...

Lee, Andrea J.

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The biophysical characterization of the three activation states of CooA through protein unfolding studies.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	The biophysical characterization of the three activation states of CooA through protein unfolding studies./
作者:	Lee, Andrea J.
面頁冊數:	186 p.
附註:	Adviser: Judith N. Burstyn.
Contained By:	Dissertation Abstracts International68-04B.
標題:	Chemistry, Biochemistry. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3261413

The biophysical characterization of the three activation states of CooA through protein unfolding studies.
Lee, Andrea J.

The biophysical characterization of the three activation states of CooA through protein unfolding studies. - 186 p.

Adviser: Judith N. Burstyn.

Thesis (Ph.D.)--The University of Wisconsin - Madison, 2007.

CooA is a transcription factor and heme-dependent CO sensor in the organism Rhodospirillum rubrum. Under anoxic conditions, CooA can be activated by CO to bind to DNA and induce transcription of a CO-metabolizing system that allows R. rubrum to live on CO as a sole energy source. CooA has three heme activation states: Fe(III), Fe(II) effector-free, and Fe(II)-CO, each of which corresponds to a distinct protein conformational state. In order to better characterize these conformations, the three activation states of CooA were unfolded using guanidine hydrochloride (GuHCl). Inactive, Fe(III) WT CooA unfolds in a complicated, multi-step process that shows the presence of one or more intermediates. Unfolding studies on CooA variants provided information about different structural regions of the Fe(III) WT CooA protein, and revealed that the domains of the protein unfold independently. The DNA-binding domain of Fe(III) WT CooA unfolds at lower concentrations of GuHCl, while the heme domain unfolds above 3 M GuHCl. Unfolding of Fe(III), Fe(II), and Fe(II)-CO WT CooA occurs with significant differences in the CD, fluorescence, and visible profiles for different protein coordination states. To confirm that the stability differences for the coordination states of WT protein are due to peptide structural changes, we also unfolded a variant of CooA, RYLLRL CooA, that is active in all heme coordination states. The CD unfolding profiles for RYLLRL CooA indicate that polypeptide stability of this variant is not dependent on heme coordination, confirming that the differences in WT CooA unfolding are due to the distinct conformations of the WT protein. A mechanism for unfolding of Fe(III), Fe(II), and Fe(II)-CO CooA was developed, which predicts that all CooA coordination states and variants undergo unfolding of the DNA-binding domain between 2 and 3 M GuHCl, while the heme domain unfolding is highly variable. These studies provide further evidence that the CooA domains are modular, and that activation occurs with changes in the position, but not structure, of the DNA-binding domain.Subjects--Topical Terms:

1017722
Chemistry, Biochemistry.

The biophysical characterization of the three activation states of CooA through protein unfolding studies.
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245 1 4 $a The biophysical characterization of the three activation states of CooA through protein unfolding studies.
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500 $a Adviser: Judith N. Burstyn.
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502 $a Thesis (Ph.D.)--The University of Wisconsin - Madison, 2007.
520 $a CooA is a transcription factor and heme-dependent CO sensor in the organism Rhodospirillum rubrum. Under anoxic conditions, CooA can be activated by CO to bind to DNA and induce transcription of a CO-metabolizing system that allows R. rubrum to live on CO as a sole energy source. CooA has three heme activation states: Fe(III), Fe(II) effector-free, and Fe(II)-CO, each of which corresponds to a distinct protein conformational state. In order to better characterize these conformations, the three activation states of CooA were unfolded using guanidine hydrochloride (GuHCl). Inactive, Fe(III) WT CooA unfolds in a complicated, multi-step process that shows the presence of one or more intermediates. Unfolding studies on CooA variants provided information about different structural regions of the Fe(III) WT CooA protein, and revealed that the domains of the protein unfold independently. The DNA-binding domain of Fe(III) WT CooA unfolds at lower concentrations of GuHCl, while the heme domain unfolds above 3 M GuHCl. Unfolding of Fe(III), Fe(II), and Fe(II)-CO WT CooA occurs with significant differences in the CD, fluorescence, and visible profiles for different protein coordination states. To confirm that the stability differences for the coordination states of WT protein are due to peptide structural changes, we also unfolded a variant of CooA, RYLLRL CooA, that is active in all heme coordination states. The CD unfolding profiles for RYLLRL CooA indicate that polypeptide stability of this variant is not dependent on heme coordination, confirming that the differences in WT CooA unfolding are due to the distinct conformations of the WT protein. A mechanism for unfolding of Fe(III), Fe(II), and Fe(II)-CO CooA was developed, which predicts that all CooA coordination states and variants undergo unfolding of the DNA-binding domain between 2 and 3 M GuHCl, while the heme domain unfolding is highly variable. These studies provide further evidence that the CooA domains are modular, and that activation occurs with changes in the position, but not structure, of the DNA-binding domain.
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