Kristoffer Chin: Week 7
From OpenWetWare
Jump to navigationJump to search
Terms
- Fabs - contains one antigen site for binding and considered a univalent antibody. Has chains linked by disulfide bonds
- Tropism - an involuntary response that can be positive or negative to the source
- Nuclear Magentic Resonance (NMR) - a way of measuring the atomic nuclei in covalent bonds through magnetic moment. NMR uses a spectroscopy type of result
- Syncitia - an epithelium or tissue with cytoplasmic continuity.
- Cyclization - chanign an open chain hydrocarbon into a closed ring
- Glycosylation - process of adding sugar units, addition of glycain chains to proteins. Carbohydrate addition to proteins
- Ramachandran Plot - a graphical representation of the angle of rotation in alpha carbons to carbonyl carbon bonds in polypeptides
- Murine - a member of the rodent family muridae
- Rhinovirus - vrius that infects the upper respiratory tract
- Glycoprotein - protein with covalent attached sugar units bonded by OH or Amine
Outline
Introduction
- Human immunodeficiency virus type 1 (HIV-1) has an outer surface embedded with spikes made up of envelope glycoproteins gp120 and gp41
- Gp41 responsible for anchoring gp120 to the outer surface
- Gp120 responsible for binding to CD4 receptors
- Gp120 has 5 variable regions
- The V3 region is integral to viral infectivity
- V3 progresses initial infection to full blown AIDS
- Interacts with coreceptors CCR5 or CXCR4
- Called principal neutralizing determinant
- Attracts naturalizing antibodies in the body
- Properties of V3 loops against vaccines
- Different exposure on V3 loop for different strains
- Neutralization resistance
- Inaccessible due to carbohydrate masking and gp120 interactions
- Adapts to different regions on its way to full blown AIDS
- V3 Conformation
- Gp120 adopts several different conformational states
- Variation in V3 region
- Aiming for recognition of antibodies on V3 loop to find a dominant structure
- Previous findings of structures
- V3 structures can be determined through fusion
- V3 insert can adjust its conformation to conform to Fabs 59.1 and 58.2
- V3 conformation to Fabs look similar and predictable through the turns it takes
- Finding a way to stabilize the conformations of V3
- Antibodies used to find the dominant conformation structure for V3 region
Results and Discussion
- Electron Density maps
- R Values were higher than expected despite great pictures
- High R values due to refinement
- Specific residues were found along each Fab molecule.
- VAL in L2 and Ser in L1
- Unpredictable results in electron desnity picture of proteins with fab interaction
- Residues make a tip from the loop that bends away from the binding sites
- loops are difficult to picture using the Electron density
- Tip shows movement away from positions
- Residues H128-H135 are disordered due to oxygen making a kink base
- There were other kinked bases that were not predicted due to their bases
- Residue Conformation
- Residues showing extended conformation making double turns that was seen in Fab 59.1 and 50.1 peptides.
- Differences in angles found, but shape remains the same
- Fab 83.1 shows some differences from 50.1 and 59.1 due to relative disposition of the residues that causes the turns
- Contact with light and heavy chains but no charge interactions.
- Analyzing the Structure
- Fab 83.1, 50.1, and 59,1 showed three peptides of similar conformation, but 58.2 shows differences from the residue tip.
- All four antibodies generated from related set of mice and the peptides were used to immunization and cocrystallization to isolate sequence
- Antibodies lacking in strength and structure and have CDR loops of different shape, size, and sequence
- Same shape but different binding orientations and locations
- These Antibodies were chosen for this experiment because of there variations for finding preferred conformations of the V3 loops
- V3 loops structures identifies show high conformation on the virus and even though this is the case, there are ways to access the virus with antibodies as shown with Fab 58.2
- Quaternary structures of the gp120 and gp41 is needed to understand how HIV-1 is able to bind to CD4 T-cells and fuse together.
Materials and Methods
- Fab Purification and Crystallizations
- Antibodies of Fab were produced from mice and purified with immobilized protein
- Used a concentration of 15 mg/ml for crystallizations studies
- Fab mixed with 16-mer peptide and crystals were gown and observed after 3 days, but analyzed over a 2-week period
- Data Collection
- Crystals cyrocooled to prevent decay
- Crystal data collected at Advanced Photon Source
- Structure Determination
- Use of Matthew's coefficient to estimate Fab molecules
- Rotation through Crowther fast rotation function
- EPMR program used to position the models over the cell and then refinement.
- Model Building and Refinement
- Measured out data and found the R Values
- Structure Analysis
- Fabs numbered by Kabat convention
- Use MS to calculate surface areas that were buried
- Hydrogen bonds analyzed by HBPLUS
- van der Waals analyzed by Contacysm
BIOL398-01: Bioinformatics Lab
- Lab Journal
- Shared Journal
- Assignments
