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Protein Structure Determination An Introduction Chad A. Brautigam, - - PowerPoint PPT Presentation
Protein Structure Determination An Introduction Chad A. Brautigam, - - PowerPoint PPT Presentation
Protein Structure Determination An Introduction Chad A. Brautigam, Ph.D. Structural Biology Laboratory UTSW Medical Center at Dallas What is Protein Structure? Arg-Lys-Ala-Gln-Trp-Cys-His-Ala-Asp What is Protein
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What is “Protein Structure?”
- It is the description of the three-
dimensional architecture of a polypeptide.
- Our aim: to know accurately and
precisely the position of every atom in a protein. That’s usually between 2000 and 10,000 atoms!
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Wherefore Art Thou, Protein Structure?
- To know a protein’s shape is to get
a glimpse of its function.
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Sometimes, Structure = Function
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Other Times, Structure Suggests Function
A structure can give us some wonderful things, i.e. testable hypotheses!
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How Do We Obtain Protein Structures?
- Nuclear Magnetic Resonance
(similar to MRI)
- Electron Crystallography
- Neutron Crystallography
- X-ray Crystallography
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In Order to Do Protein Crystallography, We Need… Protein Crystals!
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Protein Crystals
In order to obtain crystals of a protein, it is almost always necessary to have pure protein.
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Protein Sources
- Animal, plant, or microbial tissues
- r cultures.
- Companies.
- Overexpression.
– E. coli. – Insect cells. – Mammalian cell culture.
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Protein Purification
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Protein Purification
- We use the physical properties of
the protein of interest to separate it from all contaminants.
- Chief among these properties are:
– Charge. – Size.
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Quality Assessment
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Quality Assessment
Mass Spectrometry
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Now That We Have Pure Protein… How do we crystallize it?
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Protein Crystallization
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Some Common Water- Withdrawing Chemicals (Precipitants)
- Polyethylene Glycol.
- Ammonium Sulfate.
- Sodium Chloride.
- 1,4 Methyl Pentane Diol.
- Ethyl Alcohol.
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Enough Theory- How Do We Actually Do This? Vapor Diffusion!
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Vapor Diffusion
“reservoir” “Cover slip” “Drop” Crystal
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In Most Cases, Protein Crystals Are Not an End They are the means by which we can determine a protein’s structure.
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X-ray Diffraction by Crystals
X-ray Generator X-ray Detector
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An X-Ray Diffraction Pattern
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Electron Density
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Analysis of the Protein Structure
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Analysis of the Protein Structure
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