AH Biology: Unit 1 Proteomics and Protein Structure 1

1. AH Biology: Unit 1 Proteomics and Protein Structure 1 There's little doubt that proteomics -- the study of an organism's complete complement of proteins -- will have great impact in all areas of the life sciences in the years to come. And the reason is clear. "To really understand biological processes, we need to understand how proteins function in and around cells since they are the functioning units," Hanno Steen, director of the Proteomics Center at Boston Children's Hospital.

2. Think • What is the proteome? • What codes for the proteome? • How will we figure out how the proteome works? • Why is it important that we understand the proteome? • What are the applications of this technology to mankind in the future?

3. Proteomics • The proteome is the entire set of proteins expressed by a genome. • Activation and inactivation of genes • Transcription animation • Translation animation

4. RNA splicing

5. RNA splicing • When mRNA is transcribed in eukaryotic cells it is composed of introns and exons. • Introns are the non-coding sequence of the mRNA and will not be expressed in the protein molecule. They are spliced out (removed) from the mRNA. • Exons are the coding sequence and will be expressed in the protein molecule. • RNA splicing in detail.

6. Post-translational modification • Post-translational modification is the alteration of the protein after translation • Post-translational modification occurs in the rough endoplasmic reticulum, Golgi apparatus and target site of the protein. • Post-translational modification can involve • 1. the addition of chemical groups • 2. the covalent cleavage of the polypeptide

7. Post-translational modification 1. the addition of chemical groups that are catalysed by dedicated post-translational modification enzymes: • phosphorylation (addition of a phosphate group) • acylation (addition of an acyl group RCO–, where R is an alkyl group) • alkylation (addition of an alkyl group, e.g. methylation) • glycosylation (addition of a sugar group, e.g. glucose or oligosaccharides) • oxidation. 2. the covalent cleavage of sections of the polypeptide • proteases (trypsinogen to trypsin) • autocatalytic cleavage (the zymogen pepsinogen to pepsin).

8. Post-translational modification • These modifications give the proteins specific functions and target the proteins to specific areas within the cell and the whole organism. • Intracellular, e.g. lysosymes found in lysosomes and proteins required for organelles such as mitochondria. • Membrane bound, e.g. intrinsic and extrinsic proteins. • Extracellular, e.g. insulin and digestive enzymes.

9. Membrane proteins

10. Extracellular proteins and exocytosis

11. RNA splicing and post-translational modification • RNA splicing and post-translational modification results in the proteome being larger than the genome. • One gene may code for many proteins. • The proteome may be as many as three orders of magnitude larger than the genome. • Human genome = 30,000 genes approximately. • Human proteome > 100,000 proteins.

12. Regulation of gene expression • Because of regulation of gene expression not all genes are expressed as proteins in a particular cell. • The Jacob Monod hypothesis or lac Operon is an example of this process. • This ensures that the cell is energy efficient and producing proteins only when they are required.

13. the lac Operon and its control

14. Analysis of the genome • While DNA sequencing and microarray technology allow the routine analysis of the genome and transcriptome, the analysis of the proteome is far more complex. • Genome analysis involves the following techniques: • Sanger sequencing in detail • gel electrophoresis • cycle sequencing • microarray in detail.

15. Analysis of the proteome • Proteome analysis involves: • Isolation of proteins expressed by an active cell at a given time. • The functional interaction between the proteins active in the cell.

16. Analysis of the proteome • Techniques used to identify expressed proteins: • 2D electrophoresis to separate out proteins from cell samples according to their charge (isoelectric point: pH at which the protein has no net charge and does not migrate in an electric field) and molecular weight (SDS PAGE). • Western blotting: Transfer proteins to nitrocellulose paper. Expose proteins to specific antibody coupled to a radioisotope, easily detectable enzyme or fluorescent dye. Identify desired protein/proteins. • Mass spectrometry to separate out proteins and identify specific fragments.

17. Analysis of the proteome • This is a complex process as the proteins expressed differ from cell to cell and within the life cycle of the cell. • In a multicellular organism all the different cell types throughout the lifetime of the organism would have to be sampled in order to determine all the possible proteins expressed. • Proteomics technologies and cancer.

18. SDS PAGE A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

19. SDS PAGE Banding represents proteins. Smaller proteins travel further

20. Isoelectric point • Isoelectric point: pH at which the protein has no net charge and does not migrate in an electric field.

21. Western blotting Used to identify specific amino-acid sequences in proteins

22. Mass spectrometry • For more detail on mass spectrometry click the following link to Leeds University.

23. Protein structure and activity • The distinguishing feature of protein molecules is their folded nature and their ability to bind tightly and specifically to other molecules. • For example enzymes and the induced fit to their substrate • Also binding of oxygen to haemoglobin

24. Enzymes induced fit

25. Haemoglobin

26. Binding and conformational change • Binding causes a conformational change in the protein, which may result in an altered function and may be reversible. • Enzyme inhibition • Sodium potassium pump • Cell proliferation and phosphorylation • Proteins may have one or more stable conformations depending on binding. • This allows the property, regulation and activity of the protein to be controlled. • The proteasome animation.

27. Proteomics: further reading • Boston Children’s Hospital: Interactive guide to sequencing and identifying proteins. • Read the following journals to see how proteomics is used. These journals will form the basis for Proteomics Tutorials 1 and 2. • Knight JDR, Qian B, Baker D, Kothary R (2007) Conservation, Variability and the Modeling of Active Protein Kinases. PLoS ONE 2(10): e982. doi:10.1371/journal.pone.0000982. • Roy N, Nageshan RK, Pallavi R, Chakravarthy H, Chandran S, et al. (2010) Proteomics of Trypanosoma evansi Infection in Rodents. PLoS ONE 5(3): e9796. doi:10.1371/journal.pone.0009796.

28. Think • What is the proteome? • What codes for the proteome? • How will we figure out how the proteome works? • Why is it important that we understand the proteome? • What are the applications of this technology to mankind in the future?