Kristoffer Chin: Week 7

[[Media:HIV structure.ppt|Powerpoint for week 8]]

Terms

 * 1) Fabs - contains one antigen site for binding and considered a univalent antibody. Has chains linked by disulfide bonds
 * 2) *http://www.mblab.gla.ac.uk/~julian/Dict.html
 * 3) Tropism - an involuntary response that can be positive or negative to the source
 * 4) *http://www.biology-online.org/dictionary/Tropism
 * 5) Nuclear Magentic Resonance (NMR) - a way of measuring the atomic nuclei in covalent bonds through magnetic moment. NMR uses a spectroscopy type of result
 * 6) *http://www.biology-online.org/dictionary/Nuclear_magnetic_resonance
 * 7) Syncitia - an epithelium or tissue with cytoplasmic continuity.
 * 8) *http://www.biology-online.org/dictionary/Syncytium
 * 9) Cyclization - chanign an open chain hydrocarbon into a closed ring
 * 10) *http://www.biology-online.org/dictionary/Cyclization
 * 11) Glycosylation - process of adding sugar units, addition of glycain chains to proteins. Carbohydrate addition to proteins
 * 12) *http://www.biology-online.org/dictionary/Glycosylation
 * 13) Ramachandran Plot - a graphical representation of the angle of rotation in alpha carbons to carbonyl carbon bonds in polypeptides
 * 14) *http://www.biology-online.org/dictionary/Ramachandran_plot
 * 15) Murine - a member of the rodent family muridae
 * 16) *http://www.biology-online.org/dictionary/Murine
 * 17) Rhinovirus - vrius that infects the upper respiratory tract
 * 18) *http://www.biology-online.org/dictionary/Rhinovirus
 * 19) Glycoprotein - protein with covalent attached sugar units bonded by OH or Amine
 * 20) *http://www.biology-online.org/dictionary/Glycoprotein

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