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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