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

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

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?

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

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

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

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.

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.

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

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

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.

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

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

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

 SDS Electrophoresis Physics & Chemistry Comparative Proteomics Protein Profiling

Day 1 Day 2 Day 3

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?

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

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

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

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 + –

SDS-Page Example Prestained Standards Shark Salmon Trout Catfish Sturgeon Actin & Myosin Myosin Heavy Chain Actin Tropomyosin Myosin Light Chains

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

Any Questions?