Salomon Garcia: Week 7

Preparation for Journal Club 2

 * The Article for Journal Club 2; this is the link: [[Media:Sdarticle.pdf| Article for Journal Club 2]]
 * Link to the powerpoint presentation: [[Media:HIV_structure.ppt|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 http://www.biology-online.org/dictionary/Twinning
 * 2) CDR- complementarity determining region (CDR) is a relatively short amino acid sequence found in the variable (V) domains of antigen receptors http://en.wikipedia.org/wiki/Complementarity_determining_region
 * 3) Fab proteins- fragment of immunoglobulin prepared by papain treatment. http://en.wikipedia.org/wiki/Fragment_antigen_binding
 * 4) Deglycosylated-The removal of the sugar entity from a glycogen http://en.wikipedia.org/wiki/Glycosylated
 * 5) Peptide cyclization- Changing an open-chain hydrocarbon to a closed ring in a peptide bond http://www.biology-online.org/dictionary/Cyclization
 * 6) TOM/FRODO- a computer program that builds graphic images of a particular model. http://www.netsci.org/Resources/Software/Struct/frodo-tom.html
 * 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. http://en.wikipedia.org/wiki/Ramachandran_plot
 * 8) Matthews coefficient-  is used in machine learning as a measure of the quality of binary (two-class) classifications. http://en.wikipedia.org/wiki/Matthews_correlation_coefficient
 * 9) ASW mice- a hybrid mice often used in experimentation http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WFC-4DVV3KW-68&_user=945462&_coverDate=09%2F30%2F1970&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1239328754&_rerunOrigin=google&_acct=C000048964&_version=1&_urlVersion=0&_userid=945462&md5=d815f433d607ca144e09b5f405c90f23
 * 10) BALB/c mice-is an albino, laboratory-bred strain of the House Mouse from which a number of common substrains are derived. http://en.wikipedia.org/wiki/Balb/c

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.

Results and Discussion

 * Electron Density maps were good quality
 * R-values were higher due to constant refinement and adjustment