Transcript LOCALIZATION OF A MOLECULE

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?