Jordan T. Detamore Week 8
From OpenWetWare
Jump to navigationJump to search
HIV Journal Club
Definitions
- oligomeric-a polymer consisting of fewer than 20 monomers
- glycosylate-the formation of linkages with glycosyl groups.
- ectodomain-extracellular domain on membrane-anchored proteins
- immunoglobulins-any of several classes of structurally related proteins that function as antibodies or receptors and are found in plasma and other body fluids and in the membrane of certain cells.
- fusogenic-facilitate fusion
- chemokine-any of a group of cytokines produced by various cells (as at sites of inflammation) that stimulate chemotaxis in white blood cells (as neutrophils and T cells)
- isomorophous-having similar shape, form, or structure
- staves-strands or narrow strips that run between two structures to act as a covering or lining
- distal-The more (or most) distant of two (or more) things
- protomer-A structural subunit of a larger structure.
Outline
Result/Message
- The main results of this study related to the structure and mechanism of HIV-1
- A series of binding and conformational changes lead to the fusion of HIV-1 and CD4 cells
- gp120 of HIV-1 locates cells and initiates fusion
Importance/Significance
- This is an important study because the gp120 glycoprotein plays a role in receptor binding and interactions with antibodies
- Understanding this protein is important in studying HIV in order to treat the disease as well as prevent it.
Limitations in Previous Studies
- The study never discusses the limitations in other studies however it is possible that they are using newer technology in order to study the interactions of CD4 cells and gp120 of HIV
Methods
Protein production, crystallization and data collection
- Chinese hamster ovarian cells were used to acquire CD4 domains
- Monoclonal antibody 17b was isolated from HIV-1 infected host and fused with B-cell partner
- gp120 was derived from Drosophila
- Biochemical manipulations, protein purification, and ternary complex crystallization followed
- Crystals were crosslinked, equilibrated, and flash-frozen
- Data was collected using phosphor imaging plates and a Fuji BAS2000 scanner
Structure Determination and Refinement
- MERLOT was used to create Fab models
- XPLOR was used to produce a Patterson correlation
- Crystals were screened for isomorphous replacement using SCALEPACK
- Heavy atom sites were identified using Fourier analysis
- Phasing parameters were refined with MLPARE
- Density maps were made using MAPMAN
- Alpha-Carbon backbone was modeled with Program O
- Program O also aligned sequences
- PHD helped to position the amino-acid sequence
- Annealing and position refinements were simulated using XPLOR
Structure Analysis
- Deviations of CD4 structure were measured
- Significant deviations were described as having a root-mean-square residue deviation greater than the overall value and more than 0.5 angstroms greater than variation among the free structures
- Structural alignments were made using SCOP database and automatic searches were made with PrISM
Figures/Tables
- Fig. 1
- Ribbon diagram showing gp120, CD4, and Fab 17b
- Shows positioning of all components
- Fig. 2
- A-C: Different representations of gp120 from different angles
- D: Sequence alignment of HIV-1 (B, C, & O), HIV-2, and SIV
- Fig. 3
- Interactions of CD4 and gp120 shown through:
- Ribbon diagrams
- Electron density
- Electrostatic surfaces
- CD4-gp120 contact surfaces
- CD4-gp120 mutational hot-spots
- Side-Chain/Main-Chain contribution to the gp120 surface
- Sequence Variability
- Phe43 Cavity
- CD4-gp120 interface
- gp120 contacts around Phe43 and Arg 59 of CD4
- Interactions of CD4 and gp120 shown through:
- Fig. 4
- Neutralizing antibody 17b-gp120 interface shown through:
- Worm diagrams
- Contact surface and V3 loop
- Electrostatic potential
- Neutralizing antibody 17b-gp120 interface shown through:
- Fig. 5
- Processes that initiate fusion of viral and target membranes
- Table 1
- Data relating to Spacing, Molecular Replacement, Electron Density, and Refinement Statistics of Structures of Antibodies
Compare to Previous Studies
- This study provides the mechanism and further details of the aspects of HIV-1 binding that were known from other studies. Many of the discoveries from this study are additions to prior discoveries.
Powerpoint
Media:BioinfHIVJournalClub.pdf
Acknowledgements
- I worked with Isai Lopez, Colin Wikholm, and Anu Vashneyaboth in class and outside of class on Monday October 24th for this weeks electronic notebook and for the group powerpoint.
- I received instruction from Dr. Dahlquist's in Bioinformatics class
- While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.
- Jordan T. Detamore 20:17, 24 October 2016 (EDT):
References
Week 8 Assignment created by Kam D. Dahlquist
Useful links
LMU Seaver College of Science and Engineering
