2026-05-23T01:02:28Zhttps://keep.lib.asu.edu/oai/requestoai:keep.lib.asu.edu:node-1531672024-12-20T18:25:12Zoai_pmh:alloai_pmh:repo_items153167
https://hdl.handle.net/2286/R.I.27408
http://rightsstatements.org/vocab/InC/1.0/
All Rights Reserved
2014
2016-12-01T03:48:31
xvii, 132 p. : ill. (mostly col.)
Doctoral Dissertation
Academic theses
Text
eng
Gong, Zhen
Fromme, Petra
Mor, Tsafrir
Ros, Alexandra
Redding, Kevin
Arizona State University
Partial requirement for: Ph.D., Arizona State University, 2014
Includes bibliographical references (p. 106-126)
Field of study: Chemistry
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.<br/><br/>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.
Chemistry
Biogeochemistry
Biophysical characterization
Crystallography
gp41
HIV-1
Membrane proximal region
Transmembrane domain
HIV (Viruses)
Glycoproteins
Physical biochemistry
X-Ray Crystallography
Membranes (Biology)
Structural studies of the transmembrane and membrane proximal domains of HIV-1 gp41 by x-ray crystallography