structure and function of bacteriorhodopsin...
TRANSCRIPT
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Structure and Function of BacterioRhodopsin (bR)
MCDB 4810
Presented by: Matthew Bull
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Objectives
• Practice assessing the quality of x-ray and electron-beam crystallographic structure determinations
• Investigate how structure informs function
• Discuss the structure determination and refinement of a seminal membrane protein: Bacteriorhodopsin (bR)
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Overview
• Context: the accepted bR structural model
• Quick discussion of methods for investigating folding and stability of bR
• Improvement of bR structure by x-ray crystallography to 2.5Ǻ
• Refinement of bR by electron-crystallographic
means
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bR is natively in dense, periodic trimers known as purple membrane
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bR undergoes structural confirmation changes in light
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What is bR’s physiological function?
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Why is bR exciting for biophysicists?
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Questions to consider
• How can structural determination inform the role of water molecules in the proton transfer?
• Why might proton pumping out of the cell be a valuable asset?
• Why does it matter how the bR orients itself in the membrane? How is this controlled?
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Does a hydropathy plot of the 248 residues inform structure?
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AFM can probe the single-molecule stability of bR embedded in purple
membrane
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Questions to consider
• Why might trimer formation assist molecular stability?
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Lipidic cubic phase with monoolien, water and membrane protein enables
stacked crystal formation
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The model along the c-axis showed trimer lattices
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Refined model from electron beam analysis overlaid with current x-ray structure
Red: EM-Structure Green: Current Structure
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Given the differences, should we trust this analysis? Does it meet our
criteria?
Refined by molecular replacement
On average “poorly defined”
Chain continuous Sequence fits Density
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Questions to consider
• Review: How would the sampling of the diffraction pattern change if the bR unit cell spacing was increased?
• What is the difference between the AB loop and the BC loop indicate about bR? Anything significant beyond the temperature factor?
• Does this model seem reasonable? What factors draw your attention as questionable?
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A useful discussion of unique features of e-beam refinement
• High Quantum efficiency scattering
• Higher elastic/inelastic scattering ratio
• Potential for multiple scattering
• The presence of diffuse scattering backgrounds
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Discussion of a Ramachandran plot for service in refinement
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The temperature factor as a function of amino acid sequence
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Questions to consider
• How does the R_free factor improve objectivity in modeling?
• What difficulties might need to be addressed in NMR corroborating structures?
• Is bR well enough characterized that it can be used as a standard system of study to assess the accuracy of future techniques?
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Thanks for your time
• I hope you felt this discussion was useful. If you have suggestions for improving for Thursday, I would be happy to do my best to implement them. Thanks.
– Matt