Salomon Garcia: Week 7

Preparation for Journal Club 2

• The Article for Journal Club 2; this is the link: Article for Journal Club 2
• Link to the powerpoint presentation: Journal Club 2 Powerpoint

Ten New words from the 2003 Stanfield Article:

1. Twinning- The assemblage of two or more crystals, or parts of crystals, in reversed position with reference to each other in accordance with some definite law
2. CDR- complementarity determining region (CDR) is a relatively short amino acid sequence found in the variable (V) domains of antigen receptors
3. Fab proteins- fragment of immunoglobulin prepared by papain treatment.
4. Deglycosylated-The removal of the sugar entity from a glycogen
5. Peptide cyclization- Changing an open-chain hydrocarbon to a closed ring in a peptide bond
6. TOM/FRODO- a computer program that builds graphic images of a particular model.
7. Ramachandran plot- is a way to visualize dihedral angles ψ against φ of amino acid residues in protein structure.[1] It shows the possible conformations of ψ and φ angles for a polypeptide.
8. Matthews coefficient- is used in machine learning as a measure of the quality of binary (two-class) classifications.
9. ASW mice- a hybrid mice often used in experimentation
10. BALB/c mice-is an albino, laboratory-bred strain of the House Mouse from which a number of common substrains are derived.

http:wikipedia.org

http:biology-online.org/dictionary

Outline

Introduction

• HIV has an outer surface of glyco proteins
  • gp120 responsible for binding to CD4 receptors and coreceptors, CCR5 or CXCR4
  • gp41 anchors gp120 to the viral membrane providing noncovalent association

Materials and Methods

• • Mab 83.1 was made by immunization of an ASW mice with cyclic peptide RP70
  • Antibody was produced in ascites fluid of a mice and purified with an immobilized protein A column
  • Fab was made from immunoglobin by cleavage
  • Fab was concentrated to 15.0 mg/ml for crystallization studies

• Fab Purification
  • Fab was mixed with 16-mer peptide MP1 in a 6:1 mole ration
  • Crystals were grown using sitting-drop vapor diffusion method with a reservoir solution of 1.6 M Na/K phosphate, 5% isopropanol, pH 6.0.
  • Crystals grow as clusters of thin plates
  • Crystals used for this experiments grew over a 2-week period

• Fab Crystallization procedure
  • Were cryocooled to liquid nitrogen temperatures in order to collect the data in a rapid manner.
  • The crystals were protected by putting them in a solution containing the following 25% glycerol, 1.6 M Na/K phosphate, 5% isopropanol, pH 6.0
  • HKL2000 was used to format all of the data that was obtained

• Structure determination
  • Matthew coefficient was obtained by two Fab molecules
  • Model was constructed from the constant region of Fab 58.2
  • EPMR program was used to position the model in the cell
  • EPMR also used to locate the first Fab molecule in the asymetric unit

• Model building and refinement
  • TOM/FRODO was used to rebuild the mutated hybrid model and to correct the sequence and were subsequently refined with CNS version 1.1.
  • Refinement was carried with tight NCS restraints in the beginning and progressively released towards the end of refinement

• Structural Analysis
  • Kabat convention was used to number the molecules
    • Light and heavy chains are labeled using “L” and “H”
    • Peptide labeled “P” and was numbered according to HXB2 isolate sequence
  • HBPLUS was used to evaluate the Hydrogen bonds
  • Contacsym program was used to assign van der waals contacts.