國立虎尾科技大學 |

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

紀錄類型:	書目-語言資料,手稿 : Monograph/item
正題名/作者:	Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography./
作者:	Gong, Zhen.
面頁冊數:	1 online resource (150 pages)
附註:	Source: Dissertations Abstracts International, Volume: 76-06, Section: B.
Contained By:	Dissertations Abstracts International76-06B.
標題:	Biochemistry. -
電子資源:	click for full text (PQDT)
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. - 1 online resource (150 pages)

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

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

Includes bibliographical references

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.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2024

Mode of access: World Wide Web

ISBN: 9781321390438Subjects--Topical Terms:

582831
Biochemistry.
Subjects--Index Terms:

Biophysical characterizationIndex Terms--Genre/Form:

554714
Electronic books.

Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography.
LDR:04109ntm a22004337 4500 001 1145398
005 20240618081838.5
006 m o d
007 cr mn ---uuuuu
008 250605s2014 xx obm 000 0 eng d
020 $a 9781321390438
035 $a (MiAaPQ)AAI3666145
035 $a (MiAaPQ)asu:14436
035 $a AAI3666145
040 $a MiAaPQ $b eng $c MiAaPQ $d NTU
100 1 $a Gong, Zhen. $3 1470704
245 1 0 $a Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography.
264 0 $c 2014
300 $a 1 online resource (150 pages)
336 $a text $b txt $2 rdacontent
337 $a computer $b c $2 rdamedia
338 $a online resource $b cr $2 rdacarrier
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.
504 $a Includes bibliographical references
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.
533 $a Electronic reproduction. $b Ann Arbor, Mich. : $c ProQuest, $d 2024
538 $a Mode of access: World Wide Web
650 4 $a Biochemistry. $3 582831
650 4 $a Analytical chemistry. $3 1182118
650 4 $a Biogeochemistry. $3 642004
653 $a Biophysical characterization
653 $a Crystallography
653 $a Gp41
653 $a HIV-1
653 $a Membrane proximal region
653 $a Transmembrane domain
655 7 $a Electronic books. $2 local $3 554714
690 $a 0425
690 $a 0486
690 $a 0487
710 2 $a Arizona State University. $b Chemistry. $3 1186935
710 2 $a ProQuest Information and Learning Co. $3 1178819
773 0 $t Dissertations Abstracts International $g 76-06B.
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3666145 $z click for full text (PQDT)