Download Structure function

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
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
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.
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.
Pitfalls
-biological relevance. Need to be thoughtful
about interpretation. Again need to test
theories.
What are some methods for analyzing structure??
Xray crystalography
Circular dichroism (CD)
NMR
Sedimentation
Atomic force microscopy
Electronparametic spin resonance
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.
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.
Protein models
Electron density map
What you see
What you predict
End result
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
Example of how a particular structure appears in CD
Sedimentation equilibrium
Sedimentation equilibrium is an analytical ultracentrifugation method for
measuring protein molecular masses in solution and for studying proteinprotein 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).
Example of trimers using sedimentation equilibrium
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Mechanism for conformational change of influenza
hemagglutinin
Infection of cells by influenza
virus (flu)
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Model of the fusion between HA and membrane
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
Figure 1
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Model?
Other biophysics methods to address questions
Regarding proteins (conformation, size…)
Figure 1
Figure 2
Figure 3
Figure 4: Model
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