LOCALIZATION OF A MOLECULE

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LOCALIZATION OF A MOLECULE Powered By Docstoc
					Using Structure to Elucidate
   Function/Mechanism
An experimental strategy for


  – determining the arrangement of
  atoms/molecules in space to obtain
  structural information
    Some situations in which this strategy
    might be applied

–   understand a mechanism in detail
–   structure-based (rational) drug design
–   elucidate a novel structure
–   understand a conformational switch (e.g.
    prions)
Some methods that provide structural
information:
•   X ray bombardment of crystals
•   circular dichroism (CD)
•   nuclear magnetic resonance (NMR)- size < 25 kD
•   sequence comparison/analysis (e.g. COILS or
    threading)- surface residues are most conserved
•   electron microscopy (EM)
•   atomic force microscopy
•   photo/chemical cross-linking
•   protease digestion
•   calorimetry
•   sedimentation
                     Circular Dichroism
The differential absorption of radiation polarized in two directions
as a function of frequency is called dichroism. Circular dichroism
(CD) spectroscopy is a form of light absorption spectroscopy that
measures the difference in absorbance of right- and left-circularly
polarized light (rather than the commonly used absorbance of
isotropic light) by a substance.




             Kurt D. Berndt   http://broccoli.mfn.ki.se/pps_course_96/ss_960723_21.html
                 Circular Dichroism
Circular dichroism is very sensitive to the secondary
structure of polypeptides and proteins.




         Kurt D. Berndt   http://broccoli.mfn.ki.se/pps_course_96/ss_960723_21.html
What are the experimental prerequisites for
structural studies?

– material for study should be of homogeneous
  composition. How?
– often useful to reduce the complexity of the
  problem by focusing on a subregion of a larger
  molecule. How?
– minimize flexibility of the molecule in solution
  so that only a small number of conformations
  are possible. How?
What are possible pitfalls of
crystallographic studies?

– biologically irrelevant structure? Less common
  than you might think.

– faulty interpretation. Interfaces that form
  during packing of molecules into a crystal may
  not be biologically relevant. Solution?

				
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posted:12/18/2012
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