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Structural Studies of the Transmembr...

Gong, Zhen.

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Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography./
Author:	Gong, Zhen.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2014,
Description:	150 p.
Notes:	Source: Dissertations Abstracts International, Volume: 76-06, Section: B.
Contained By:	Dissertations Abstracts International76-06B.
Subject:	Biogeochemistry. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3666145
ISBN:	9781321390438

Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography.
Gong, Zhen.

Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography. - Ann Arbor : ProQuest Dissertations & Theses, 2014 - 150 p.

Source: Dissertations Abstracts International, Volume: 76-06, Section: B.

Thesis (Ph.D.)--Arizona State University, 2014.

This item is not available from ProQuest Dissertations & Theses.

The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution. This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 A after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.

ISBN: 9781321390438Subjects--Topical Terms:

545717
Biogeochemistry.
Subjects--Index Terms:

Biophysical characterization

Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography.
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245 1 0 $a Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2014
300 $a 150 p.
500 $a Source: Dissertations Abstracts International, Volume: 76-06, Section: B.
500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Fromme, Petra;Mor, Tsafrir.
502 $a Thesis (Ph.D.)--Arizona State University, 2014.
506 $a This item is not available from ProQuest Dissertations & Theses.
506 $a This item must not be sold to any third party vendors.
520 $a The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution. This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 A after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
590 $a School code: 0010.
650 4 $a Biogeochemistry. $3 545717
650 4 $a Analytical chemistry. $3 3168300
650 4 $a Biochemistry. $3 518028
653 $a Biophysical characterization
653 $a Crystallography
653 $a Gp41
653 $a HIV-1
653 $a Membrane proximal region
653 $a Transmembrane domain
690 $a 0425
690 $a 0486
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710 2 $a Arizona State University. $b Chemistry. $3 2096519
773 0 $t Dissertations Abstracts International $g 76-06B.
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792 $a 2014
793 $a English
856 4 0 $u https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3666145