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Towards membrane protein structure d...

Savage, David F.

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Towards membrane protein structure determination.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Towards membrane protein structure determination./
作者:	Savage, David F.
面頁冊數:	217 p.
附註:	Adviser: Robert M. Stroud.
Contained By:	Dissertation Abstracts International68-02B.
標題:	Biophysics, General. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3251931

Towards membrane protein structure determination.
Savage, David F.

Towards membrane protein structure determination. - 217 p.

Adviser: Robert M. Stroud.

Thesis (Ph.D.)--University of California, San Francisco, 2007.

Membrane protein structure determination, as well as functional characterization, has significantly lagged behind similar investigations of soluble proteins. Membrane proteins are typically expressed at low levels, must be solubilized from the membrane in detergents, and even when purified often heterogenous. Thus, membrane proteins present many unique challenges. To overcome these challenges we have selected a model membrane protein, AqpZ, of the aquaporin family of water channels. Such a model system can be used not only to investigate the structure-function properties of this class of channels, but also to elucidate fundamental conventions which can be applied to all membrane proteins. We have expressed, purified, and solved the x-ray crystal structure of AqpZ. To determine the mechanism of water selectivity a conserved region of the channel was engineered to allow conduction of other substrates and functional analysis shows the basis to be chemical rather than steric in nature. Furthermore, aquaporins were characterized with the use of mercurial compounds, and using AqpZ we have also elucidated a steric inhibition mechanism by mercurials. From these and other studies, it became apparent that protein expression was a fundamental barrier to working with membrane protein so we therefore set out to develop new expression systems and protocols targeted towards membrane proteins. We have adapted a reconstituted cell-free protein expression system that can express proteins at the milligram level and shown these proteins can be solubilized and purified to homogeneity. Finally, with an eye towards structural genomics we have introduced a series of protocols to streamline structure determination. As a test case the structure of the monotopic membrane protein CcmG was solved using these protocols with a minium amount of inputed work. Thus we have demonstrated, from both a single protein and a structural genomics level, a unified approach for moving towards membrane protein structure determination.Subjects--Topical Terms:

1019105
Biophysics, General.

Towards membrane protein structure determination.
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502 $a Thesis (Ph.D.)--University of California, San Francisco, 2007.
520 $a Membrane protein structure determination, as well as functional characterization, has significantly lagged behind similar investigations of soluble proteins. Membrane proteins are typically expressed at low levels, must be solubilized from the membrane in detergents, and even when purified often heterogenous. Thus, membrane proteins present many unique challenges. To overcome these challenges we have selected a model membrane protein, AqpZ, of the aquaporin family of water channels. Such a model system can be used not only to investigate the structure-function properties of this class of channels, but also to elucidate fundamental conventions which can be applied to all membrane proteins. We have expressed, purified, and solved the x-ray crystal structure of AqpZ. To determine the mechanism of water selectivity a conserved region of the channel was engineered to allow conduction of other substrates and functional analysis shows the basis to be chemical rather than steric in nature. Furthermore, aquaporins were characterized with the use of mercurial compounds, and using AqpZ we have also elucidated a steric inhibition mechanism by mercurials. From these and other studies, it became apparent that protein expression was a fundamental barrier to working with membrane protein so we therefore set out to develop new expression systems and protocols targeted towards membrane proteins. We have adapted a reconstituted cell-free protein expression system that can express proteins at the milligram level and shown these proteins can be solubilized and purified to homogeneity. Finally, with an eye towards structural genomics we have introduced a series of protocols to streamline structure determination. As a test case the structure of the monotopic membrane protein CcmG was solved using these protocols with a minium amount of inputed work. Thus we have demonstrated, from both a single protein and a structural genomics level, a unified approach for moving towards membrane protein structure determination.
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