Isaiah M. Castaneda Week 7

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BIOL368/F11:Week_7

Journal Club 2 - Stanfield et. al 2003

Contents

Outline of Stanfield et. al Article, 2003

I. HIV-1: A lentivirus, surface contains envelope glycoproteins gp120 & gp41

  • Those 2 proteins are encoded by gp160 polypeptide chain
  • Gp41 attaches itself to membrane
  • Gp120 is noncovalently associated around gp41
—Responsible for binding to CD4 receptor and others
—5 variable regions
•V1-V5
  • V3
—40 residues of differing length
—Particularly important
—Variation linked to several HIV-1 aspects
—Peptides with V3 region tended to bring to neutralizing antibodies
—Interest in creating vaccine using this region
—However, they have trouble neutralizing early isolates

Later in development, either there are conformational changes or gp120 becomes less stable

  • Gp120

After binding to CD4 cells, gp120 undergoes many conformational changes

  • Accordingly, V3 shape & exposure also undergoes changes

Aim to answer how V3 loop recognition and change affects HIV-1 virus

  • Structure analysis of peptides with V3 loops from various HIV-1 isolates
—Antibodies 50.1 & 59.1 recognize 1 V3 conformation
—Antibody 58.2 recognizes another

Conformations differ at loop around GPGRAF sequence (highly conserved) Beta strand followed by Beta turn around GPGRAF is common to both conformations V3 inserted into VP1 protein from rhinovirus type 14

  • Virus still binds to 59.1 and 58.2 Fabs
—Insert conformed to very different antibody sites
  • NMR studies
—When bound to antibody, peptide forms a loop
—V3 peptides lack order in water
•Beta turns around GPGR region
—20% TFE, cyclization, aminoisobutyric acid, glycolysation
•Stabilize V3 loop

II. Antibody 83.1

  • Has an exceptional neutralization trait
—Neutralized broad range of viral strains

Results of study with Fab 83.1

  • Negative tests for twinning
High R-value = noncrystallographic translation
—R close to integer, strong
—R close to half integer, weak
  • Used crystollagraphic data to construct Fab 83.1-V3 complexes

It is seen that L1 loop bends away from its usual position to avoid clashing with other residues Complementary Determining Region H3 is kinked, which was unexpected

  • H93 – Ala
  • H94 – Ile
  • H101 – Asp

Normally a kinked base is the result of Asp forming a salt bridge with Arg or Lys (charge AA’s) Near charged N-terminus, but does not interact with it

  • Bound to a water molecule instead
  • Two other Fabs have unpredicted kinked bases
•Fab 26-10
H93 – Ala
H94 – Gly
H101 – Asp
Hydrogen bonded to H27 (Tyr)
•Fab B02C11
H93 – Ala
H94 – Val
H101 - Asp
Hydrogen bonded to nitrogens of H96 (Asp) & H99 (Ala)

Used electron density maps to ensure that carbonyl oxygens were in the right place
V3 peptide has multiple contacts with Fab 83.1 ¾ of the peptides that have strong neutralization properties share a similar shape

  • Although shapes are similar, they are very different otherwise
—Sites at which V3-peptide binds is different

III. Materials/Methods

  • Created 83.1 peptide by immunizing mice with RP70
  • Anitbodies were purified
  • Produced and concentrated Fab fragments
  • Mixed them with a MP1 peptide
  • Allowed crystals to grow for 2 weeks on thin plates
  • To protect data, crystals were quickly immerse into a solution
  • Examined peaks using Patterson map, rotations using Crowser function, then constructed a model
  • EPMR program used to further improve their model
  • Further refined their models with more programs
  • Used MS program to calculate buried molecular surface areas
  • Used HBPLUS and Contacsym programs to assess hydrogen bondsand Van Der Waal’s interactions

Definitions

As I investigated the article, I immediately noted words/abbreviations that were very foreign to me. The list below ensued. The majority of the definitions were found online, but the last two were harder to find from a reputable source, so their definitions were taken from the Oxford Dictionary of Biochemistry & Molecular Biology. I have the e-book.

  1. Lentivirus - Exogenous horizontally and vertically transmitted viruses found in humans and other mammals; associated with immunodeficiencies, neurological disorders, and arthritis.
    http://de.dict.md/definition/lentivirus. Oct 16, 2011
  2. Tropism - An involuntary orienting response; positive or negative reaction to a stimulus source.
    http://www.biology-online.org/dictionary/Tropism. Oct 12, 2011
  3. Rhinovirus - picornaviridae that largely infect the upper respiratory tract. Include the common cold virus and foot and mouth disease virus.
    http://www.biology-online.org/dictionary/Rhinovirus. Oct 12, 2011
  4. Chimera - Organism composed of two genetically distinct types of cells. Can be formed by the fusion of two early blastula stage embryos or by the reconstitution of the bone marrow in an irradiated recipient or by somatic segregation.
    http://www.biology-online.org/dictionary/Chimeras. Oct 16, 2011
  5. Fab - Part of an immunoglobulin antibody that binds a specific antigen and consists of both a light chain and part of a heavy chain. By comparison, natural antibodies consist of two heavy and two light chains. An FAB (fragment antibody) offers the advantages of smaller size and lower cross-reactivity compared to the complete antibody.
    http://de.dict.md/definition/FAB. Oct 16, 2011
  6. TFE - 2,2,2-trifluoroethanol, CF 3CH2OH. A protein denaturant, because it weakens hydrophobic interactions.
    http://what-when-how.com/molecular-biology/trifluoroethanol-molecular-biology/. Oct 16, 2011
  7. Cyclization - Changing an open-chain hydrocarbon to a closed ring.
    http://www.biology-online.org/dictionary/Cyclization. Oct 16, 2011
  8. 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; also, rarely, in artificial twinning (accomplished for example by pressure), the process by which this reversal is brought about. Polysynthetic twinning, repeated twinning of crystal lamellae, as that of the triclinic feldspars. Repeated twinning, twinning of more than two crystals, or parts of crystals.
    http://www.biology-online.org/dictionary/Twinning. Oct 16, 2011
  9. Canonical - An archetypical nucleotide or amino acid sequence to which all variants are compared.
    "canonical sequence." Oxford Dictionary of Biochemistry and Molecular Biology. New York: Oxford University Press Inc, 2000. 91.
  10. Ascites - the accumulation of serous fluid in the peritoneal cavity. The presence of ascites may be due to the growth of a tumour elsewhere in the body, the ascitic fluid then containing a suspension of single tumour cells. Administration of a hybridoma results in the formation of monoclonal antibodies which can be recovered from the ascitic fluid.
    "ascites." Oxford Dictionary of Biochemistry and Molecular Biology. New York: Oxford University Press Inc, 2000. 51.
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