Protein Profiling

Background Information

How do we determine evolutionary relationships between organisms?

Proteomics The study of protein structure and function This includes: Protein-protein interactions Protein expression Posttranslational modifications of protein-coding genes Proteome = all proteins in a particular cell

How are proteins made?

Posttranscriptional Modifications: the creation of proteins RNA Editing Alternative Splicing mRNA Synthesis & Degradation Proteolytic Cleavage Protein Degradation Protein-Protein Interaction Carbohydrate Modification (Glycosylation) Phosphorylation

1. 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

2. Alternative Splicing Alternative splicing allows exons to be included or excluded to produce different mRNA’s This of course leads to the production of different proteins Example:

3. 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.

4. Proteolytic Cleavage Most proteins undergo cleavage after translation: Peptide bonds are broken via protease Usually ‘met’ is removed Misfolded proteins can also undergo proteolytic cleavage

5. Protein Degradation Very similar to mRNA degradation Ubiquitin: The molecular kiss of death! Proteins degraded by proteasomes

6. 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. For example:

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

8. Phosphorylation The addition of a PO4 group to a protein by a kinase Activates or deactivates by causing a conformational change For example: Smooth muscle contracts when phosphorylated

Hopefully we can see that it becomes increasingly important to examine protein expression & modification among species…

Our Experiment

Day 1: Extract Fish muscle proteins

What is Laemmli buffer? What does it do? Contains SDS, Tris buffer, Glycerol, and Bromophenol blue Denatures and solubilizes proteins

What is DTT? What does it do? DTT stands for Dithiothreitol It is a reducing agent that breaks the proteins disulfide bonds

Day 2: Run an SDS-PAGE Gel

What does SDS-PAGE stand for?

What is polyacrylamide? Gel matrix is polyacrylamide – a polysaccharide Smaller pores to separate small biomolecules Proteins are much smaller than DNA Average amino acid = 110 daltons Average nucleotide pair = 649 daltons

What are the two layers of the gel? What is their purpose?

Gel Mechanisms A molecule’s mobility through gel is affected by 2 things: Mass Charge

What charge do proteins have? Proteins can have +, - or Ø charge So how do we fix this problem? SDS!

What is SDS? SDS detergent (sodium dodecyl sulfate) - CH2 CH3 SDS SDS detergent (sodium dodecyl sulfate) Solubilizes and denatures proteins Adds negative charge to proteins s-s SDS, heat Proteins with SDS + –

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

Actin and Myosin Standard Used as a reference to help ID major conserved muscle proteins & serves as a control

Day 3: Analyze Proteins

Procedure

Tips These gels are ONE MILLIMETER thick. They are very fragile once out of the casing. Loading them will be tricky, and you will need to use the “protein only” pipette tips.

Results

Your Gel Will Show Proteins of Many Sizes kD Function Titin 3000 Center myosin in sarcomere Dystrophin 400 Anchoring to plasma membrane Filamin 270 Cross-link filaments Myosin heavy chain 210 Slide filaments Spectrin 265 Attach filaments to plasma membrane Nebulin 107 Regulate actin assembly -actinin 100 Bundle filaments Gelosin 90 Fragment filaments Fimbrin 68 Actin 42 Form filaments Tropomysin 35 Strengthen filaments light chain 15-25 Troponin (T.I.C.) 30, 19, 17 Mediate contraction Thymosin 5 Sequester actin monomers 10 15 20 25 37 50 75 100 150 250 Measure distance from base of wells to the base of the bands Measure fish protein bands between ~30 and 10 kD Measure prestained standard bands between ~30 and 10 kD Prestained Standards A B C D E Actin & Myosin