Ryan N. Willhite Week 7: Difference between revisions
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==Outline== | |||
* Entry of HIV involves a sequential interaction of the envelope glycoprotein (gp120) | I. Introduction | ||
* | |||
* | *Entry of HIV involves a sequential interaction of | ||
* | **the envelope glycoprotein (gp120) | ||
* Entry is mediated by envelope glycoproteins | **CD4 glycoprotein | ||
* 5 variable regions | **chemokine receptor (primary receptor) | ||
* Variable and non variable regions are glycosylated | *CD4i (antibodies that block gp120-CD4 complexes to the chemokine receptor) | ||
*CCR5 and CXCR4 for HIV-1 (secondary receptors) | |||
*CD4 binding induces conformational changes in the gp120 | |||
*Entry of HIV is mediated by envelope glycoproteins | |||
*5 variable regions | |||
* V3 loop determines specificity | *Variable and non variable regions are glycosylated | ||
*V3 loop determines specificity | |||
* Gp41 (transmembrane coat proteins) variants found in all enveloped viruses | *Gp41 (transmembrane coat proteins) variants found in all enveloped viruses | ||
* | *N-terminal fusion peptides which participate in membrane fusion | ||
* Enveloped viruses tend to be | *Enveloped viruses tend to be characteristic in entry | ||
*Direct membrane penetration (HIV) | |||
* | *HIV causes destruction of CD4 T cells which ultimately leads to AIDS. | ||
Methods | II. Purpose of this Study | ||
*Gp120 glycoprotein has important role in | |||
**receptor binding | |||
** interactions with neutralizing antibodies | |||
* Information about the gp120 structure is important for understanding HIV infection | |||
*Assist in designing therapeutic strategies. | |||
*Overall purpose is to observe the mechanism of HIV entry and intervene | |||
III. Determining the Structure | |||
*Devised a crystallization strategy that modified the protein surface | |||
*Obtained crystals of | |||
**a ternary complex composed of a truncated form of gp120 | |||
** the N-terminal two domains (DID2) of CD4 | |||
**Fab from the human neutralizing monoclonal antibody 17 | |||
*The ternary structure was solved by | |||
**combinations of molecular replacement | |||
** isomorphous replacement | |||
** density modification techniques | |||
IV. Methods | |||
* Protein Production, crystallization and data collection | * Protein Production, crystallization and data collection | ||
Diffraction data were collected at beamline X4A using phosphorimaging plates and Fuji BAS2000 scanner | Diffraction data were collected at beamline X4A using phosphorimaging plates and Fuji BAS2000 scanner | ||
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** Automatic concatenation of unmodelled density using PRISM | ** Automatic concatenation of unmodelled density using PRISM | ||
* Measure deviation | * Measure deviation | ||
[[Media:JOURNALCLUB.ppt|Powerpoint Presentation]] | |||
==Unfamiliar Words: == | |||
#glycoprotein-are proteins that contain oligosaccharide chains covalently bound to polypeptide side chains. | |||
#chemokine-One of a large group of proteins that act as lures and were first found attracting white blood cells. | |||
#cavity-laden- | |||
#oligomeric-A polymer that consists of two, three, or four monomers. | |||
#virion-A complete viral particle, consisting of RNA or DNA surrounded by a protein shell and constituting the infective form of a virus. | |||
#fusogenic-cause cell fusion | |||
#interfacial cavities | |||
#haemagglutinin-involved in influenza. clogs red blood cells together. | |||
#steric occlusion- as a General Pro Region Inhibition Mechanism | |||
*pubs.acs.org/cgi-bin/jtext?bichaw/36/i13/abs/bi962341o | |||
#cryoprotectant- protects biological tissue from freezing. | |||
*[[BIOL398-01/S10:Week 7|Week 7 Assignment Page]] |
Latest revision as of 00:53, 8 March 2010
Outline
I. Introduction
- Entry of HIV involves a sequential interaction of
- the envelope glycoprotein (gp120)
- CD4 glycoprotein
- chemokine receptor (primary receptor)
- CD4i (antibodies that block gp120-CD4 complexes to the chemokine receptor)
- CCR5 and CXCR4 for HIV-1 (secondary receptors)
- CD4 binding induces conformational changes in the gp120
- Entry of HIV is mediated by envelope glycoproteins
- 5 variable regions
- Variable and non variable regions are glycosylated
- V3 loop determines specificity
- Gp41 (transmembrane coat proteins) variants found in all enveloped viruses
- N-terminal fusion peptides which participate in membrane fusion
- Enveloped viruses tend to be characteristic in entry
- Direct membrane penetration (HIV)
- HIV causes destruction of CD4 T cells which ultimately leads to AIDS.
II. Purpose of this Study
- Gp120 glycoprotein has important role in
- receptor binding
- interactions with neutralizing antibodies
- Information about the gp120 structure is important for understanding HIV infection
- Assist in designing therapeutic strategies.
- Overall purpose is to observe the mechanism of HIV entry and intervene
III. Determining the Structure
- Devised a crystallization strategy that modified the protein surface
- Obtained crystals of
- a ternary complex composed of a truncated form of gp120
- the N-terminal two domains (DID2) of CD4
- Fab from the human neutralizing monoclonal antibody 17
- The ternary structure was solved by
- combinations of molecular replacement
- isomorphous replacement
- density modification techniques
IV. Methods
- Protein Production, crystallization and data collection
Diffraction data were collected at beamline X4A using phosphorimaging plates and Fuji BAS2000 scanner
- Structure determination
- To locate Fab 17b in the ternary complex crystals, rotational searches with 52 different Fab models were made with program MERLOT*
- Crystals soaked in over 20 different heavy atom compounds
- Isomorphous replacement phasing*
- Automatic concatenation of unmodelled density using PRISM
- Measure deviation
Unfamiliar Words:
- glycoprotein-are proteins that contain oligosaccharide chains covalently bound to polypeptide side chains.
- chemokine-One of a large group of proteins that act as lures and were first found attracting white blood cells.
- cavity-laden-
- oligomeric-A polymer that consists of two, three, or four monomers.
- virion-A complete viral particle, consisting of RNA or DNA surrounded by a protein shell and constituting the infective form of a virus.
- fusogenic-cause cell fusion
- interfacial cavities
- haemagglutinin-involved in influenza. clogs red blood cells together.
- steric occlusion- as a General Pro Region Inhibition Mechanism
- pubs.acs.org/cgi-bin/jtext?bichaw/36/i13/abs/bi962341o
- cryoprotectant- protects biological tissue from freezing.