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Protein Structure Determination
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Drug Design Techniques
TARGET structure

• "Small molecule" Modelling
• Superimposition of known ligands
• Conformational analysis
• Pharmacophore hypotheses
• 3D-Database searches

• Structure-based Design
• Search for "hot spots"
• Pharmacophore hypotheses
• 3D-Database searches
• Docking
• De-novo Design

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How to get 3D structures of proteins

• Experimentally
• Xray Crystallography
• Multidimensional NMR spectroscopy
• Cryo-Electron Microscopy
• Computationally
• Homology Modelling

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Principle of Xray Crystallography

• Protein crystals are grown from aqueous solutions
  by varying conditions
• Temperature
• Salt concentration
• pH
• Crystals are placed into an intense Xray beam
• Xrays are scattered (diffracted) by electrons of
  the protein
• Diffraction pattern is collected
• Electron density is calculated by Fourier
  transformation
• 3D model is constructed and refined

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Xray Crystallography Protein Crystals
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Xray Crystallography Hardware
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Xray Crystallography Diffraction
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Xray Crystallography Diffraction
Diffraction only occurs when conditions of
Bragg's Law are obeyed
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Xray Crystallography From Diffraction to
Electron Density Maps
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Xray Crystallography From Density Maps to 3D
models
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Xray Crystallography Resolution
gt Higher resolution of Xray diffraction yields
3D models of higher quality
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Xray Crystallography Resolution
gtResolution can be increased by lowering the
temperature
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Xray Crystallography Important terms
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Xray Crystallography Example
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Principle of Xray Crystallography

• Protein crystals are grown from aqueous solutions
  by varying conditions
• Crystals are placed into an intense Xray beam
• Xrays are scattered (diffracted) by electrons of
  the protein
• Diffraction pattern is collected
• Electron density is calculated by Fourier
  transformation
• 3D model is constructed and refined
• Structures of Protein-Ligand complexes can be
  determined either by co-crystallisation or by
  soaking

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Principle of NMR Spectroscopy

• NMR spectrum of a protein is measured in solution
• Structural information is derived, based on
• Nuclear Overhauser Effect (NOE) -gt atom-atom
  distances
• J-Coupling constants -gt torsion angles
• From distance and angle constraints, a 3D model
  can be calculated by Molecular Dynamics
  simulations

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NMR Spectroscopy Basics
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NMR Spectroscopy Basics

• A second magnetic field is applied at varying
  frequencies
• Resonance absorbtion of nuclear spins can be
  measured
• Resonance absorbtion depends on atom type and
  atomic environment ("chemical shift")
• Resonance transfer across non-covalent atom
  pairscan be measured ("Nuclear Overhauser
  Effect")

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NMR Spectroscopy Nuclear Overhauser Effect
Distances of non-covalent atomscan be measured
as cross-peaksin 2D NMR spectra Peak intensity
can be related todistance of the atom
pair Resulting NOE distances canbe applied as
constraints in Molecular Dynamics calculations
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NMR Spectroscopy Example
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Cryo-Electron Microscopy

• Uses electron beams instead of xrays
• Is well suited for structure determination of
  2-dimensional crystals
• Is currently the only technique to determine
  high-resolution structures of transmembrane
  proteins

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Cryo-Electron Microscopy
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Cryo-Electron Microscopy Example Frog Rhodopsin
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Homology Modelling

• Goal Generation of a 3D protein model from
  sequence only
• Steps
• Find homologues proteins with known structures,
  e.g. from PDB (www.rcsb.org/pdb)
• Align sequences of structurally known proteins
• Find structurally conserved regions
• Align with target sequence (structurally unknown
  protein)
• Construct structurally conserved regions (SCRs)
• Construct structurally variable regions (SVRs)
• Run Molecular Dynamics simulation to refine model

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Homology Modelling Sequence Alignment
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Homology Modelling

• Summary
• Works well for sequences with high homology to
  structurally known protein (gt30 identity) and
  when several members of the family are known
• Structurally variable regions ("loops") cause
  problems when loops have different lengths
• Correct prediction of amino acid sidechains can
  be very difficult