Transcript Class details

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Enzyme inhibition types
Inhibitors and kinetic parameters
Paper 2
Crystallization VS Precipitation
Why crystallize proteins?
chemwiki.ucdavis.edu
Protein crystallization
http://www.nature.com/nprot/journal/v4/n5/fig_tab/nprot.2009.27_F5.html
Protein crystallization
http://www.nature.com/nmeth/journal/v5/n2/fig_tab/nmeth.f.203_F2.html
Protein crystallization
http://www.nature.com/nmeth/journal/v5/n2/fig_tab/nmeth.f.203_F2.html
Sensitivity of protein crystallization
http://www.dolomite-microfluidics.com/centeo_web/index.php?option=com_content&view=article&id=212:protein-crystallization-theory&catid=2:design&Itemid=398
How to get crystals NOT Ppt?
Start off with undersaturated
Very slowly move to supersaturated
Equilibrate to allow nucleation & crystallization
Every protein different!
Art VS Science!
Protein crystallization
http://www.nature.com/nmeth/journal/v5/n2/fig_tab/nmeth.f.203_F2.html
Factors affecting protein crystallization
Vaporization to equilibrate
Lysozyme facts
Second protein structure
First protein sequenced with all 20 amino acids
First enzyme mechanism
Lysozyme in eggs…
http://digital.nls.uk/scientists/biographies/alexander-fleming/discoveries.html
…and body fluids (eg. mucous)
http://digital.nls.uk/scientists/biographies/alexander-fleming/discoveries.html
Lysozyme is defense mechanism
PLoS ONE 9(2): e89130. doi:10.1371/journal.pone.0089130
Breaks bonds in peptidoglycan layer
G+ve VS G-ve
Lysozyme action
Bacteria
Lysozyme
Water
Heat + Alkaline lysis
Measure cell lysis
The Journal of Infectious Diseases Vol. 96, No. 1 (Jan. - Feb., 1955), pp. 75-81
Lab
Make egg extracts (get the egg white!)
Total protein in egg extract
Enzyme activity (OD of Micrococcus luteus)
Set up crystallization trays with purified lysozyme & egg extract