Structure Determination and Globular Proteins

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Structure Determination and Globular Proteins
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X-ray crystallography

• Visible light is approx. 4000 Angstroms and the
  average covalent bond distance is 1.5 Angstroms.
  Not very practical for obtaining a structure.
• X-rays are approximately 1.5 Angstroms and thus
  are used.
• However, there is no such thing as x-ray
  microscope and focus in on an image.
• X-ray diffraction patterns, generated by atoms in
  a regular crystal lattice is recorded (x-ray
  film) and then are used to construct the
  structure.
• Intensity of each spot is function of electron
  density

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X-ray crystallography

• From diffraction patterns, an electron density
  map is generated. With this map, atoms are
  fitted. Shown above is the x ray diffraction
  pattern, the electron density map, the heme of
  myoglobin fitted in and the rest of the protein.
• When primary sequence is known, task is much
  easier, almost necessary.
• Portion of the human rhinovirus protein is shown
  to the left.

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X-ray crystallography

• Generating crystals still challenging.
  Presently, robotics and high through put methods.
  Crystals can be colored due to chromophors in
  proteins.
• Proteins crystals still mostly water (60).
  Thus, the soft consistency limits the resolution
  down to 2 Angstroms.
• Proteins in crystals are catalytically active.
  Thus this proves that conformation of protein is
  native.

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2D-NMR

• Nuclear magnetic resonance can also provide
  structures of proteins (lt20 kd).
• Monitor NMR signal of protons in proteins. Apply
  large external magnetic field which separates
  energies of nuclear spin (with or against field).
  Input of radiofrequency flips spin from lower to
  higher energy state.
• Energy levels also influenced by spin of
  neighboring protons (mini magnets rather than
  large external magnets). The inter-atomic
  distance, if less than 0.5 Angstroms give rise to
  this coupling or nuclear Overhauser effect
  (NOESY). Coupling can also occur through bonds
  as determined by correlated spectroscopy (COSY).

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2-D NMR

• The off diagonal peaks or cross peaks arise from
  interation of two protons that are lt 5 Angstroms
  apart and whose 1D-NMR peaks are located where
  the horizontal and vertical lines through the
  cross peak intersects the diagonal.
• Knowing the identify of these peaks allows the
  molecule to be assembled in space.
• Inter-proton distance measurements are not
  precise. Thus structure is ensemble of closely
  related structures.
• Structures obtained from NMR and X ray are
  typically in good agreement.

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Visualizing proteins

• 3-D structures of many proteins available.
• Many sites available for downloading coordinates.
• http//www.rcsb.org/pdb/
• http//www4.ncbi.nlm.nih.gov/entrez/query.fcgi
• Once downloaded, structures can be viewed by a
  number of free software.
• http//www.umass.edu/microbio/rasmol/
• http//us.expasy.org/spdbv/
• A number of viewing options available where
  elements of secondary structure are shown. Can
  also combine with details of side chains.

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Lignin Peroxidase

• Stick view where all atoms except hydrogen are
  shown

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Lignin Peroxidase

• Line view where only peptide backbone is shown.

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Lignin Peroxidase

• Ribbon view where secondary structures are
  colored.

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Lignin Peroxidase

• Schematic view where secondary structures are
  colored.

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Lignin Peroxidase

• Schematic view also showing the heme (green).

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Tendency to hide hydrophobic residues

• Yellow portion shows hydrophobic sections of
  proteins.

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Topology diagram

• Visible light is approx.

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Hairpin b motifs
Bovine trypsin inhibitor is shown in a. Hairpin
b motif is in red. Snake venom erabutoxin is
shown in b and has hairpin b motif shown in red
and green.

• Common but not associated with function
• This motif is two adjacent antiparallel strands
  joined by a loop. Called either a hairpin or a
  b-b unit
• Observed as ribbons or as part of more extended
  sheets
• Length of loop connecting strands varies from 2
  to 5 amino acids

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b-a-b motif

• Connection between strands of parallel b-sheets
  are frequently made by a-helix. Thus motif has
  b-strand, loop, a-helix, loop and b-strand
• Found in almost all proteins that have parallel
  b-sheets
• Helical axis usually parallel with that of
  b-sheet
• Helix packs against sheet thus shielding
  hydrophobic residues of b-sheet
• The loop regions can vary in length. Role of
  loop connecting C end of strand to a-helix is
  often binding site or active site
• Every b-a-b motif is right handed

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a-a motifsFour helix bundles

• The simplest and most frequently found a-helical
  domain consists of 4 a helices in a bundle.
  Residues contacting each other in the bundle are
  hydrophobic.

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The globin fold

• Globin fold found in myoglobin, hemoglobin and
  phycocyanins (light capture assembly).
• Bundle of 8 a helices, A-H forms a pocket for the
  active site, heme for oxygen binding
• Protects heme from contact with other heme

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b barrels

• Structure is superoxide dismutase (SOD).
• Has 8 antiparallel b strands, also Cu and Zn
• The b strands arranged around barrel.

• Diverse group
• 4 to over 10 antiparallel strands
• typically 2 b sheets that pack against each other
• Sheets have twist and form barrel-like structure

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a-a structure
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b-a-b structures