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Last class Enzyme inhibition types Inhibitors and kinetic parameters Paper 2.

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Presentation on theme: "Last class Enzyme inhibition types Inhibitors and kinetic parameters Paper 2."— Presentation transcript:

1 Last class Enzyme inhibition types Inhibitors and kinetic parameters Paper 2

2 Crystallization VS Precipitation

3 Why crystallize proteins? chemwiki.ucdavis.edu

4 Protein crystallization http://www.nature.com/nprot/journal/v4/n5/fig_tab/nprot.2009.27_F5.html

5 Protein crystallization http://www.nature.com/nmeth/journal/v5/n2/fig_tab/nmeth.f.203_F2.html

6 Protein crystallization http://www.nature.com/nmeth/journal/v5/n2/fig_tab/nmeth.f.203_F2.html

7 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

8 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!

9 Protein crystallization http://www.nature.com/nmeth/journal/v5/n2/fig_tab/nmeth.f.203_F2.html

10 Factors affecting protein crystallization pH

11 Vaporization to equilibrate

12 Lysozyme facts Second protein structure First protein sequenced with all 20 amino acids First enzyme mechanism

13 Lysozyme in eggs… http://digital.nls.uk/scientists/biographies/alexander-fleming/discoveries.html

14 …and body fluids (eg. mucous) http://digital.nls.uk/scientists/biographies/alexander-fleming/discoveries.html

15 Lysozyme is defense mechanism PLoS ONE 9(2): e89130. doi:10.1371/journal.pone.0089130

16 Breaks bonds in peptidoglycan layer

17 G+ve VS G-ve

18 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

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