Kristoffer Chin: Week 7

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Powerpoint for week 8

Contents

Terms

  1. Fabs - contains one antigen site for binding and considered a univalent antibody. Has chains linked by disulfide bonds
  2. Tropism - an involuntary response that can be positive or negative to the source
  3. Nuclear Magentic Resonance (NMR) - a way of measuring the atomic nuclei in covalent bonds through magnetic moment. NMR uses a spectroscopy type of result
  4. Syncitia - an epithelium or tissue with cytoplasmic continuity.
  5. Cyclization - chanign an open chain hydrocarbon into a closed ring
  6. Glycosylation - process of adding sugar units, addition of glycain chains to proteins. Carbohydrate addition to proteins
  7. Ramachandran Plot - a graphical representation of the angle of rotation in alpha carbons to carbonyl carbon bonds in polypeptides
  8. Murine - a member of the rodent family muridae
  9. Rhinovirus - vrius that infects the upper respiratory tract
  10. 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
Kristoffer Chin: Week 2 Kristoffer Chin: Week 6 Kristoffer Chin: Week 11
Kristoffer Chin: Week 3 Kristoffer Chin: Week 7 Kristoffer Chin: Week 12
Kristoffer Chin: Week 4 Kristoffer Chin: Week 8 Kristoffer Chin: Week 13
Kristoffer Chin: Week 5 Kristoffer Chin: Week 9 Kristoffer Chin: Week 14


  • Shared Journal
BIOL398-01/S10:Class Journal Week 2 BIOL398-01/S10:Class Journal Week 6 BIOL398-01/S10:Class Journal Week 11
BIOL398-01/S10:Class Journal Week 3 BIOL398-01/S10:Class Journal Week 7 BIOL398-01/S10:Class Journal Week 12
BIOL398-01/S10:Class Journal Week 4 BIOL398-01/S10:Class Journal Week 8 BIOL398-01/S10:Class Journal Week 13
BIOL398-01/S10:Class Journal Week 5 BIOL398-01/S10:Class Journal Week 9 BIOL398-01/S10:Class Journal Week 14


  • Assignments
BIOL398-01/S10:Week 2 BIOL398-01/S10:Week 6 BIOL398-01/S10:Week 11
BIOL398-01/S10:Week 3 BIOL398-01/S10:Week 7 BIOL398-01/S10:Week 12
BIOL398-01/S10:Week 4 BIOL398-01/S10:Week 8 BIOL398-01/S10:Week 13
BIOL398-01/S10:Week 5 BIOL398-01/S10:Week 9 BIOL398-01/S10:Week 14

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