Lecture 19 - X RAY & NMR

Lecture 19 - X RAY & NMR - Determining Protein...

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Determining Protein Structure BIBC 100
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Determining Protein Structure X-Ray Diffraction Interactions of x-rays  with electrons  in  molecules in a crystal NMR- Nuclear Magnetic Resonance Interactions of magnetic field (external with the intrinsic  magnetic properties of  atomic nuclei which possess a spin  angular  momentum
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Determining Protein Structure X-RAY Protein Crystal “Virtually” any size  Few, high quality  crystals Result: Electron-density  map   atomic model Requires: Phase  determination of the  diffracted beams for  which heavy metals are  used NMR Spectroscopy Limited (2009) ~ 30,000  Da. Protein in High Conc. Result: Distance  constraints between  1 atoms   3-D molecular  model Requires: Isotopes ( 1 H,  13 C,  15 N) d
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Determining Protein Structure X-RAY Frozen Structure NMR Time Resolved “Dynamic”   Folding BOTH OF THEM NEED : Purified Protein Amino Acid Sequence Computers: Molecular Mechanics Molecular Dynamics Molecular Graphics Recombinant DNA Technology
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Crystals 1. Well-Ordered 2. Large Size (~ 0.5 mm) 3. “Pure” protein at high concentration HOW: Solution  Aggregate                    Reducing Protein           Solubility Amorphous Ordered  Crystal
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Crystals Methods Hanging-Drop:  Increase Conc.  By vapor diffusion Microdialysis:  Increase  aggregation by  loss of solvent Variables pH Temperature Gravity [protein] solvent
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Requirements Crystals
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This note was uploaded on 01/07/2011 for the course BIBC BIBC 100 taught by Professor Buehler during the Fall '09 term at UCSD.

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Lecture 19 - X RAY & NMR - Determining Protein...

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