Methods of determination

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• Methods of determination
• of protein structure
• by
• Ivo Frébort

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Architecture of Proteins

• Shape - globular or fibrous
• The levels of protein structure
• - Primary - sequence
• - Secondary - local structures - H-bonds
• - Tertiary - overall 3-dimensional shape
• - Quaternary - subunit organization

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What forces determine the structure?

• Primary structure - determined by
  
  covalent bonds
• Secondary, Tertiary, Quaternary structures - all
  determined by weak forces
• Weak forces - H-bonds, ionic interactions, van
  der Waals interactions, hydrophobic interactions

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Other Chemical Groups in Proteins

• Proteins may be "conjugated" with other chemical
  groups
• If the non-amino acid part of the protein is
  important to its function, it is called a
  prosthetic group.
• Glycoprotein, lipoprotein, nucleoprotein,
  phosphoprotein, metalloprotein, hemoprotein,
  flavoprotein.

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Peptide bond
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Peptide bond planes
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Steric Constraints on phi psi

• Unfavorable orbital overlap precludes some
  combinations of phi and psi
• phi 180, psi 0 is unfavorable
• phi 0, psi 0 is unfavorable
• phi 0, psi 180 is unfavorable

• G. N. Ramachandran was the first to demonstrate
  the convenience of plotting phi,psi combinations
  from known protein structures
• The sterically favorable combinations are the
  basis for preferred secondary structures

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Ramachandrans plot
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Classes of Secondary Structure

• All these are local structures that are
  stabilized by hydrogen bonds
• Alpha helix
• Other helices
• Beta sheet (composed of "beta strands")
• Tight turns
• Beta bulge

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The Alpha Helix

• First proposed by Linus Pauling and Robert Corey
  in 1951
• Identified in keratin by Max Perutz
• A ubiquitous component of proteins
• Stabilized by H-bonds

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The Beta-Pleated Sheet

• Composed of beta strands
• Also first postulated by Pauling and Corey, 1951
• Strands may be parallel or antiparallel
• Rise per residue
• 3.47 Angstroms for antiparallel strands
• 3.25 Angstroms for parallel strands
• Each strand of a beta sheet may be pictured as a
  helix with two residues per turn

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The Beta Turn

• allows the peptide chain to reverse direction
• carbonyl C of one residue is H-bonded to the
  amide proton of a residue three residues away
• proline and glycine are prevalent in beta turns

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What are the structural and functional advantages
driving quaternary association?

• Stability reduction of surface to volume ratio
• Genetic economy and efficiency
• Bringing catalytic sites together
• Cooperativity

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MAX FERDINAND PERUTZ 1962 Nobel Laureate in
Chemistry for their studies of the structures
of globular proteins. Born 1914 Place of
Birth Vienna, Austria Residence Great
Britain Affiliation Laboratory of Molecular
Biology, Cambridge Died February 6, 2002
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Braggs law
2d.sin? n.?
Fourier transform the mathematical relationship
between the electron density and the diffraction
by X-rays where F(h) is the reflection at
reciprocal lattice point h and f(x) is the
scattering function of the electron density at
point x. F(h) is a complex number (a vector)
containing the amplitude and the phase of the
reflection. The integration is over the complete
unit cell. Thus every atom contributes to the
amplitude and phase of each individual
reflection, but to varying extents. This means
that a partial data set still gives information
about the complete structure. There is also an
inverse Fourier transform in which the
electron density at each point rho(x) is made up
of a sum of all of the reflection amplitudes and
phases. Again, note that each point in the
electron density contains contributions from all
of the reflections. To get the best electron
density at each location within the unit cell a
complete data set is necessary.
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Protein crystal diffraction pattern
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Only amplitude can be measured !!! Phase solving
by heavy atom isomorphous replacement
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Hampton research - Crystal screen kits and
accessories
Hanging drop
Sitting drop
Microdialysis
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Crystal screen kit reagents
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Microbatch Crystallization
Oryx 6 Robot for microbatch and vapor diffusion
crystallization
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7 GeV
8 GeV
SPring-8
APS
ESRF
Super Photon Ring-8 GeV Harima Science Garden
City Japan
Advanced Photon Source Argonne, USA
6 GeV
European Synchrotron Radiation
Facility Grenoble, France
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SPring-8
The linac accelerates electrons generated by an
electron gun to 1 GeV using high frequency
electric fields.
The synchrotron is a circular accelerator that
accelerates electrons injected from the linac to
an energy of 8 GeV and transfers them to the
storage ring.
Electrons with an energy of 8 GeV are stored and
synchrotron radiation is produced in the storage
ring. The beamlines guide synchrotron radiation
to the experimental hutch, where scientific
research is performed.
This facility is located at the end of 1 km-long
beamline. It is used for research on advanced
coherent X-ray optics. The observation of the
gravitational effect on light is an example of
the experiments carried out there.
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Steps of protein crystallography
Density modification
Modelling
Isomorphous replacement
Refinement
Molecular replacement
Phasing
Structure
Publication
Validation
Data processing
Databases
Purification
Data collection
Crystallization
X-ray
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Crystals of copper amine oxidase from Aspergillus
niger
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Pea seedling AO
Escherichia coli AO (Parsons et al., 1995)
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Active site of pea AO
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Protein structure determination by NMR
spectroscopy
Triple resonance experiments
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R Riek, S Hornemann, G Wider, M Billeter, R
Glockshuber K Wuethrich NMR structure of the
mouse prion protein domain PrP(121-231). Nature
382, 180-182 (1996).
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Literature Garett, R. and Grisham, C.
Biochemistry 2nd ed., Harcourt Brace Company,
Orlando, FL, USA 1999. Jones, C., Mulloy, B.,
and Sanderson, M. R. Crystallographic methods
and protocols, Methods in Molecular Biology Vol.
56, Humana Press, Totowa, NJ, USA 1996. Spring-8
web page (www.spring8.jp) and other Internet
resources