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| Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography | |
| [null] | |
| 出版年 | 2014 |
| 学位授予单位 | Gong, Zhen (Author), Fromme, Petra (Advisor), Mor, Tsafrir (Advisor), Ros, Alexandra (Committee member), Redding, Kevin (Committee member), Arizona State University (Publisher) |
| 英文摘要 | abstract: 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 Å 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. Dissertation/Thesis Doctoral Dissertation Chemistry 2014 |
| 英文关键词 | Chemistry Biogeochemistry Biophysical characterization Crystallography Gp41 HIV-1 Membrane proximal region Transmembrane domain |
| 语种 | 英语 |
| URL | http://hdl.handle.net/2286/R.I.27408 |
| 来源机构 | Arizona State University |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/248358 |
| 推荐引用方式 GB/T 7714 | [null]. Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray Crystallography[D]. Gong, Zhen (Author), Fromme, Petra (Advisor), Mor, Tsafrir (Advisor), Ros, Alexandra (Committee member), Redding, Kevin (Committee member), Arizona State University (Publisher),2014. |
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