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Learn how to utilize experimental strategies to elucidate the structure and function of molecules. Discover various methods such as X-ray crystallography, NMR spectroscopy, and circular dichroism to investigate molecular structures in detail. Delve into applications like rational drug design and conformational switches. Understand the importance of homogeneous sample composition and minimizing molecular flexibility in structural studies.
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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. http://broccoli.mfn.ki.se/pps_course_96/ss_960723_21.html Kurt D. Berndt
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
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?