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					  Lecture 12


Protein Structure
                Overview

•   Classification of amino acids
•   Protein structure organization
•   Membrane protein
•   Protein structure analysis
               Proteins

Proteins are single, un-branched chains of
  amino acid with diverse functions:
  vtransport and storage
  vmotion
  vmechanical support
  vregulation
  vhost/pathogen interaction
  vcatalysis
                   Amino Acid
• Basic structural unit of a protein
• Joined by a peptide bond
• Made up of:
      • Amino group
      • Carboxyl group
      • Side chain
• Avg. MW = 113
• Least common: Cys, Met, Trp (~5% of total)
• Most common: Leu, Ser, Gly, Glu (~32% of
  total)
• Handy rule: YFP MW = # AAs X 110 da
The peptide bond



           Peptide bonds are
            rigid and planar.
Structural components of a
           protein
     Amino acid classification

• Polar
  – Basic
  – Acidic
  – Uncharged
• Nonpolar
Hydrophilic amino acids
Hydrophobic amino acids
Special Amino Acids
        Four levels of structural
              organization
• Primary

• Secondary

• Tertiary

• Quaternary
         Four levels of structural
               organization
• Primary: the linear sequence of amino acids.

• Secondary: the localized organization of parts of
  a polypeptide chain (e.g., the a helix or b sheet).

• Tertiary: the overall, three-dimensional
  arrangement of the polypeptide chain.

• Quaternary: the association of two or more
  polypeptides into a multi-subunit complex.
Secondary structure: the a helix
• Single polypeptide chain. Twists around
  itself every 3.6aa to form a rigid cylinder
• Hydrogen bond (made on every fourth
  peptide bond) linking N-H and C=O
• Distance between two turns 0.54nm
• R groups extend outward
• Abundant in portions of membrane
  proteins crossing the membrane
Secondary structure: the a helix




                *Proline and glycine break helices
Secondary structure: the b strands
• Makes up the core of many protein
  – Parallel chain: two peptide strands running in the
    same direction held together by hydrogen bonds
  – Anti-parallel chain: two peptide strands running in
    opposite directions held together by hydrogen bonds


• b Barrel- many beta strands
  – Found on outer membrane of mitochondria,
    chloroplast and many bacteria
  – act as transport proteins, receptors or enzymes
                The beta sheet




Anti-parallel              Parallel
Primary and secondary structure of
    Influenza Neuraminidase
                         Alpha
                         Helix
 Beta Sheet
                    Tertiary Structure

• Determined by a variety of
  bonding interactions
  between the "side chains" on
  the amino acids:

   –   Hydrogen bonds
   –   Disulfide bonds
   –   Non-polar interaction
   –   Salt bridges
                                    E Obayashi et al. Nature 2008
             Quaternary structure

• Globular:
  – Folded in a globular shape
  – Same or different types of            hemoglobin
    secondary structure
  – Example: haemoglobin


• Fibrous:
  – Polypeptides arranged in long
    strands or sheets                 Collagen

  – One type of secondary structure
  – Examples: collagen and silk
        Protein Domains:

• Sub-structure produced by a part of
  polypeptide chain that can fold
  independently into a compact, stable
  structure
• 40-350aa
• Associated with different
  functions
                   Regulatory domain
                            Motifs

• Signatures of protein
  families
• Used as a tool for the
  prediction protein function          zinc finger motif
   – AA sequence- functional motifs
      • zinc finger motif
   – Secondary structures-structural
     motifs
      • Helix loop helix


                                        Helix loop helix
Membrane proteins in a lipid bilayer
Membrane proteins associate with
 the lipid bilayer in different ways
   Integral membrane proteins
• They are amphipathic
• interact extensively with hydrocarbon
  chains of membrane lipids
• span lipid bilayer (single pass Vs
  multipass a helix)
• b barrel (e.g porins)
 Peripheral membrane proteins
• Attached to lipid bilayer covalently
   –   fatty acid chains: Myristatyl anchor- N terminal glycine
   –   prenyl group- C terminal cysteine
   –   palmitic acid- internal cysteine
   –   oligosaccharide (GPI anchors)- can be cleaved by
       phosholipase C
• Noncovalent interactions with other membrane
  proteins, many times integral proteins
• Can be displaced by changes in ionic strength or
  pH
Lipid anchors
 Membrane proteins can be solubilzed
     and purified in detergents
• Membrane proteins are displaced from bilayer
  with the use of detergents
• Detergents are amphipathic
• Sodium dodecyl Sulfate (SDS)
     • strong Ionic detergent
     • Solubilize hydrophobic membrane proteins
     • Denature proteins by binding to hydrophobic cores
• Triton
     • Mild non-ionic, detergent
     • proteins maintain some activity and can be purified in
       complexes
Solubilizing membrane proteins
        with a detergent
  Triton-X100        SDS
    Protein Structure Analysis

• Technique of choice
  depends on your
  needs.
     Protein Structure Analysis

• Limited proteolysis

• X-ray crystallography

• NMR

• Circular dichroism (CD)
       X-ray crystallography
• Beams of x-rays passed through a crystal
  of purified protein.


• Can be done with:
  – protein
  – protein/DNA
  – protein/RNA
  – protein/small molecule
             X-ray crystallography
•   X-ray wavelengths are short - resolves atoms (0.1nm)
•   When X-ray beam is applied on a crystal, most of it will pass through
    but a small fraction will be scattered by atoms in the sample.
•   In a well ordered crystal, the scattered waves reinforce each other
    and appear as diffraction spots
•   Diffraction patterns are analyzed into a 3D electron density map
•   3D electron density maps, with knowledge of amino acid sequence,
    can be used to determine a 3D protein structure.
Diffraction pattern
               X-ray crystallography
Issues:
•   Need gram quantities of protein.
•   NEED CRYSTAL.
•   Need heavy metal version.
•   Flexible regions don’t form structure.
•   Membrane proteins very difficult.
       NMR = Nuclear Magnetic
            Resonance
• Concentrated protein sample placed on a strong
  magnetic field
• NMR measures nuclear magnetism or changes in
  nuclear magnetism in a molecule
• Atomic nuclei of hydrogen atoms exhibit magnetic
  spin which aligns to a strong magnetic field
• Magnetic spin misalign when radio-frequency
  pulses are applied
• Radio-frequency radiation is emitted when H
  return to aligned state-measured and recoded on
  a spectrum
       NMR = Nuclear Magnetic
            Resonance
• Behavior of any atom is influenced by
  neighboring atoms – lead to spin-spin coupling
• More closely spaced residues are more
  perturbed than distant residues
• Can calculate distances based on perturbation
• Combining NMR information with aa sequences
  makes it possible to compute a 3D structure of a
  protein.
An idealized NMR spectrum




 Number of peaks=n+1
 n=number of nearby H’s
NMR spectrum of a protein




                                     2D NMR can be used
                                     to resolve Peaks


    Kurt Wüthrich Nobel prize 2002
      NMR = nuclear magnetic
           resonance

• Don’t need crystals

• Limited to proteins less than 20 kD

• OK for domains from larger proteins.
Structure-based drug discovery
• Depends on 3D structure-both solved and
  predicted

• Drug design
    • catalytic sites
    • Protein-Protein interaction

				
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posted:7/19/2013
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