Structure to Elucidate Mechanism

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• Structure to Elucidate Mechanism
• Detailed structural information can give
  unexpected insights.
• Classic example the elucidation of the structure
  of DNA by Watson and Crick (1952). The structure
  immediately suggested a means by which DNA could
  be replicated in a way that maintained its
  informational content

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• When is it appropriate to analyze the structure
  of a molecule?
• to understand a mechanism in greater detail
• Structure based drug design
• Novel structure
• Conformational switches

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• Historically structure determination performed
  late in the process of elucidating the function
  of a molecule. That may be changing.
• reliable means of predicting 3D structures from
  primary sequence?
• - structural genomics initiative to find the
  structures of large numbers of proteins.

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• Experimental prerequisites for obtaining a
  structure!
• Need a purified molecule often in abundance.
• Proteolysis helps to define a domain and then can
  overexpress in heterologous system and purify or
  can synthesize if segment is small enough. Helps
  to have sufficient knowledge of molecule to know
  how to produce a fragment on which to focus
  efforts.
• Need a lack of flexibility. For a crystal to
  form, must have a small number of conformations
  so that solution can get aggregation of similar
  conformations to grow crystal.

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Pitfalls -biological relevance. Need to be
thoughtful about interpretation. Again need to
test theories.
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What are some methods for analyzing structure??
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Xray crystalography Circular dichroism (CD) NMR
Sedimentation Atomic force microscopy Electronpar
ametic spin resonance
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XRAY Crystallography Identifies chrstalline
phases present in solid materials. A beam of
Xrays are used to bombard a specimen from various
angles. The Xrays are diffracted as they are
reflected from successive planes formed by the
crystal lattice of the material. By varying
angles a diffraction pattern emerges that is
characteristic of the samples. It can then be
further characterized by comparing with databases
of other patterns. Heavy atoms with more
electrons scattern X-rays more strongly.
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Different methods for Xray crystallography
MIR multiple isomorphous replacement Depends
upon the select binding of heavy metals
(mercury/platnum) to a limit number of sites in
the crystal. Heavy atoms scatter xrays more
strongly than light atoms they replace
MAD multiwavelength anomalous diffraction.
Replacement of methionine with selenomethionine
by overexpressing the protein in bacteria grown
in defined media containing selenomethionine. The
selenium atom replaces a sulfur in the methionine
side chain. Unlike larger metals used for MIR
these selenium atoms are tolerated well by most
proteins, crystallization tends to proceed the
same as native protein. The selenium is readily
detected by x-ray diffraction.
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Protein models
Electron density map
What you predict
End result
What you see
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Circular Dichroism Spectroscopy
Circular dichroism (CD) spectroscopy measures
differences in the absorption of left-handed
polarized light versus right-handed polarized
light which arise due to structural asymmetry.
The absence of regular structure results in
zero CD intensity, while an ordered structure
results in a spectrum. Circular dichroism
spectroscopy is particularly good
for -determining whether a protein is folded,
and if so characterizing its secondary
structure, tertiary structure, and the
structural family to which it belongs -comparing
the structures for different mutants of the same
protein -studying the conformational stability
of a protein under stress -- thermal stability,
pH stability, and stability to denaturants. I
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Example of how a particular structure appears in
CD
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Sedimentation equilibrium
Sedimentation equilibrium is an analytical
ultracentrifugation method  for measuring protein
molecular masses in solution and for studying
protein-protein interactions. Sample spun in
ultracentrifuge to force the protein toward
outside of rotor, but not high enough to cause
the sample to pellet. As centrifugal force
produces a gradient in protein concentration,
diffusion acts to oppose this concentration
gradient. An exact balance is reached between
sedimentation and diffusion and the concentration
distribution reaches equilibrium. This
equilibrium concentration is then measured while
the sample is spinning using absorbance
detection. It is particularly valuable
for -establishing whether the native state of a
protein is a monomer, dimer, trimer,
etc. -measuring the stoichiometry of complexes
between two or more different proteins or between
a protein and a non-protein ligand -measuring the
equilibrium constants for reversible
protein-protein and protein-ligand interactions
(Kd).
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Example of trimers using sedimentation
equilibrium
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Mechanism for conformational change of influenza
hemagglutinin
Infection of cells by influenza virus (flu)
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Model of the fusion between HA and membrane
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Tubby
Tub identified by positional cloning in obese mice
Tub -/- display degeneration of retina, hearing
loss
TUBLP (Tubby like protein) in human mutation
Retinitis pigmentosa
Molecular role unknown
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Model?
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Other biophysics methods to address
questions Regarding proteins (conformation, size)
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Figure 1
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Figure 2
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Figure 3
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Figure 4 Model
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Figure 5
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Figure 6
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Figure 7