using structure to elucidate function mechanism
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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

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Presentation Transcript
slide2

An experimental strategy for

  • determining the arrangement of atoms/molecules in space to obtain structural information
slide3

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)
slide4

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
slide5

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.

http://broccoli.mfn.ki.se/pps_course_96/ss_960723_21.html

Kurt D. Berndt

slide6

Circular Dichroism

Circular dichroism is very sensitive to the secondary structure of polypeptides and proteins.

http://broccoli.mfn.ki.se/pps_course_96/ss_960723_21.html

Kurt D. Berndt

slide8

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?
slide9

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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