W4:Introduction to Studying Protein

• Virtually all biotechnology products involve amino acids or proteins
• Genetically modified plants express new proteins
• Genetically modified plants

  • Pest resistance
  • Improved characteristics
• Genetically modified animals express useful proteins
• Genetically modified animal proteins

  • Antithrombin (anti blood clotting protein)
• Gene therapy aims to correct defective protein production
• Types of amino acids found in proteins

  • Charged
  • Polar (hydrophilic)
  • Non-polar (hydrophobic)

  
• Amino acid structure

  • Central carbon (α-carbon)
  • Carboxyl group (COO-)
  • Amino group (NH2+)
  • Side chain (R-group)
• Amino acid R-groups

  • Determine amino acid properties
• Amino acid R-group types

  • Charged (-COO- or -NH2+ functional groups)
  • Polar (hydrophilic) (-OH, -SH, -CONH2)
  • Non-polar (hydrophobic) (aliphatic -CH2- or aromatic group)
• Polar R-groups

  Exposed to the aqueous solvent on the outer surface of the protein
• Charged R-groups

  Neutralized in the interior (+ve opposite -ve) or exposed to the solvent where they can bind ions or other charged molecules (charge-charge interaction)
• Non-polar R-groups

  Predominantly in interior
• Protein folding

  • Determined by its shape

  
• Levels of protein structure

  • Primary structure (linear sequence of amino acids)
  • Secondary structure (areas of localized folding)
  • Tertiary structure (3D structure of complete protein)
  • Quaternary structure (overall shape formed by multiple subunit proteins)

  
• Glycoproteins

  Made up of polypeptide and carbohydrate residues, often displayed on cell surface, generally involved in recognition and binding

  
• HIV-1 uses its glycoprotein (gp120) on its envelope to attach to cells displaying CD4 and CCR5 surface receptors, facilitating viral entry into the cell

  
• Antibodies

  Identify and neutralize foreign molecules (antigens), remove from body, have capacity to identify thousands of foreign invaders
• Antibody structure

  • Made up of 4 polypeptides (2 light and 2 heavy chains linked by disulfide bridges), have constant and variable regions, flexible "hinge" allows for greater flexibility of recognizing shapes

  
• Antibody function

  They function by key-lock recognition of invaders and signal the immune system to remember the invasive party, the part of the antibody that recognizes the epitope (surface feature) of an antigen is called the paratope

  
• Antibody applications

  • Diagnostic kits (pregnancy tests, virus detection, breast cancer)
  • Forensic analysis (anthrax, blood typing)
• Monoclonal and polyclonal antibodies

  Monoclonal antibodies recognize a single antigen, polyclonal antibodies recognize several antigens
• Until recently, proteins could only be made in cells, now small polypeptide chains can be synthesized in the laboratory but it is slow and inefficient
• Protein synthesis

  1. Transcription (DNA to mRNA)

  2. Translation (mRNA to polypeptide chain)
• Transcription

  RNA polymerase binds to DNA, DNA helicase unwinds double-stranded DNA, RNA polymerase transcribes bases from one strand of DNA into pre-mRNA
• Eukaryotic post-transcriptional modification

  Pre-mRNA contains exons (sense portions) and introns (non-sense portions), modification enzymes remove introns and splice exons together, 5' cap and poly(A) tail added to form functional mature mRNA
• Translation

  mRNA associates with ribosomes in the cytoplasm, amino acids picked up by tRNA, amino acids linked to form polypeptide chain
• The genetic code is universal - bacteria can "read" eukaryotic genes and produce eukaryotic proteins, with rare exceptions like mitochondrial genes and some microorganisms/viruses
• Mitochondrial genes

  Some microorganisms, virus
• Translation

  1. 22

  2. Transcription

  3. Copy genetic instructions from DNA to RNA

  4. RNA polymerase binds to DNA

  5. DNA helicase enzyme unwinds ds DNA

  6. RNA polymerase transcribes bases from 1 strand of DNA into pre-mRNA
• Eukaryote - Post transcriptional modification

  • Pre-mRNA contains exons -"sense" portions, introns -"non-sense portions" must be removed
  • Modification enzymes remove introns and splice exons together
  • 5' Cap
  • Poly (A) tail
  • Functional mature mRNA leaves nucleus to be translated
• Translation

  1. mRNA molecule moves from nucleus to cytoplasm

  2. Becomes associated with ribosomes

  3. Ribosomes composed of ribosomal RNA, rRNA

  4. Amino acids picked up in cytoplasm by transfer RNA, tRNA

  5. Amino acids linked to form polypeptide chain
• Transfer RNA (tRNA)

  • 61 codons
  • 1 tRNA can recognize > 1 codon (wobble)
  • ~ 31 tRNAs in human
  • Each tRNA recognizes: i) its appropriate amino acid, ii) the codon on the mRNA which specifies that amino acid
  • tRNAs transfer amino acids to the mRNA on a ribosome
  • Align them in the correct order for protein synthesis as specified by the sequence of codons in that mRNA molecule
• Translation

  1. Reading of an mRNA sequence into an aa seq by tRNAs and ribosomes

  2. Ribosome is a scaffold on which translation can take place: the amino acids in their correct order are linked to form the complete protein

  3. mRNA molecules also contain the "start" and "stop" codons: thus the beginning and endpoints of the amino acid sequence are precisely defined
• Ribosome

  • Contains two sites to bind two tRNAs next to each other (A site, P site) and one site for exit (E site)
  • Attaches to mRNA, and slides along mRNA until the start codon is reached
  • Start codon (AUG) begins protein synthesis
  • A tRNA with the correct anticodon binds to the start codon on the ribosome
  • tRNA bound to its relevant amino acid - activated
• Translation

  1. Initiation: Small ribosomal subunit binds to mRNA, Initiator tRNA base pairs with start codon, Large subunit completes initiation complex

  2. Elongation: a) Anticodon of arriving aminoacyl tRNA base pairs with mRNA codon in "A" site, b) A peptide bond created between the amino acid in the "A" site and the polypeptide in the "P" site, c) tRNA in the A site is translocated by the ribosome to the "P" site; tRNA in "P" site is translocated to "E" site and released

  3. Termination: a) ribosome comes to a stop codon, accepts a protein named release factor, b) Release factor hydrolyzes the bond between tRNA and the chain in the P site, releasing the polypeptide, c) All components disassemble
• Polyribosomes

  • Cluster of ribosomes reading an mRNA molecule at once
  • Many ribosomes can read the same message at once
  • Rapidly make proteins, at different stages, along the mRNA
• There are thousands of different proteins in the body
• Each cell type contains a specific set (types and amounts) of proteins
• Cellular proteins are specified by genes