1.  Understanding the basics  SDS Electrophoresis Physics & Chemistry Comparative Proteomics Protein Profiling

2. Can biomolecular evidence be used to determine evolutionary relationships?  Changes in DNA lead to proteins with: Different functions Novel traits Positive, negative, or no effects  Genetic diversity provides pool for natural selection = evolution  Traits are the result of: Structure Function  Proteins determine structure and function  DNA codes for proteins that confer traits

3. Proteomics  Proteins are diverse! There are many modification systems that allow 1 gene to code for many proteins. Why do C. elegans & humans have ~ the same number of genes?

4. Remember Protein Structure????? 4o4o 3o3o 2o2o 1o1o

5. Posttranscriptional Modifications  RNA Editing  Alternative Splicing  mRNA Synthesis & Degradation  Proteolytic Cleavage  Protein Degradation  Protein-Protein Interaction  Carbohydrate Modification (Glycosylation)  Phosphorylation

6. RNA Editing  Evolved eukaryotes can change the sequence of mRNA’s by substituting bases.  Less evolved eukaryotes can delete bases.  This clearly changes the codons and corresponding amino acids. New stop codons New ORF’s New proteins

7. Alternative Splicing  mRNA of higher eukaryotes has 2 types of sequence segments Introns & Exons  Alternative splicing allows exons to be included or excluded to produce different mRNA’s…  This of course leads to the production of different proteins.

8. mRNA Synthesis & Degradation  Level of mRNA synthesis partly determines the level of protein expression  Chemical modifications to mRNA’s can change their stability and therefore, protein expression levels.

9. Proteolytic Cleavage  Amino acid encoded by AUG (start codon)  Most proteins undergo cleavage after translation Peptide bonds are broken via protease Usually ‘met’ is removed  Misfolded proteins can also undergo proteolytic cleavage

10. Protein Degradation  Very similar to mRNA degradation  Proteins have a ‘shelf- life’ Tagged with ubiquitin Degraded by proteasomes  These are large multienzyme complexes that break down proteins Ubiquitin – the molecular kiss of death

11. Protein-Protein Interactions  Proteins usually function in complexes. If one protein, necessary for the complex to work properly, is not produced then you may have a non-functional structure.

12. Carbohydrate Modification  Lymphocyte (T, B and NK cells) carbohydrates are essential in determining how they will infiltrate sites of infection  Many proteins in the PM are covalently bonded to carbohydrates (sugars)  Modifications to these carbs can alter how the protein functions

13. Phosphorylation  The addition of a PO 4 group to a protein by a kinase Activates or deactivates by causing a conformational change  Smooth muscle contracts when phosphorylated

14. Hopefully we can see that it becomes increasingly important to examine protein expression & modification among species… That’s what you are doing in lab!

16.  SDS Electrophoresis Physics & Chemistry Comparative Proteomics Protein Profiling

17. Day 1 Day 2 Day 3

18. SDS Electrophoresis Physics & Chemistry  SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis Used to examine proteins  How many proteins?  Molecular weight?  What differences are there in proteins from different sources?

19. Why SDS-PAGE instead of Agarose?  Gel matrix is polyacrylamide Smaller pores Separates small biomolecules  The gel is not uniform in density

20. How is Protein Size Measured?  Size measured in kilodaltons (kD)  Dalton = approximately the mass of one hydrogen atom or 1.66 x 10-24 gram  Average amino acid = 110 daltons General chemistry of an amino acid.

21. Does Charge Matter?  A molecule’s mobility through gel is affected by: 1) charge, 2) mass  Proteins can have +, - or Ø charge…So…

22. SDS to the Rescue!  SDS detergent (sodium dodecyl sulfate) Solubilizes and denatures proteins Adds negative charge to proteins O S O O O - CH 2 CH 3 SDS s-s SDS, heat Proteins with SDS + –

23. SDS-Page Example Prestained Standards Shark Salmon Trout Catfish Sturgeon Actin & Myosin Myosin Heavy Chain Actin Tropomyosin 10 15 20 25 37 50 75 100 150 250 Myosin Light Chains

25. Reagents you are working with… 1. Molecular weight marker 2. Laemmli buffer – solubilizes proteins 3. Actin & myosin standard – used as a reference to help ID major conserved muscle proteins & serves as a control 4. DTT – reducing agent that breaks the proteins disulfide bonds 5. Coomassie Stain – will use to stain proteins

26. Any Questions?