Chapter 5: Introduction to Studying Protein

1. Chapter 5: Introduction to Studying Protein Introduction to Biotechnology, BIOL1414 Austin Community College, Biotechnology Dept

2. Learning Outcomes • Describe the structure of proteins, including the significance of amino acid R-groups and their impact on the three-dimensional structure of proteins. • Explain the steps of transcription and translation in protein synthesis. • Discuss the role of naturally occurring proteins and recombinant proteins in biotechnology. • Differentiate proteins that function as part of structure, as antibodies, and as enzymes. • Describe the structure of antibodies and explain the relationship between antibodies and antigens. • Discriminate among the classes of enzymes and discuss the effect of reaction conditions on enzyme activity. • Summarize polyacrylamide gel electrophoresis and identify its usefulness for studying proteins. Note about this PowerPoint – There are several links in this PPT that allow you to explore more into different topics. Some of these links are animations, movies, or exercises. Please note, you must be in the slide show to activate the links. You can press F5 any time to active the slide show and “Esc” to exit.

3. Central Dogma • DNA codes for RNA which codes for proteins that confer traits. • Click to see Video Animation

4. The Importance of Proteins in Biotechnology • The ability to synthesize and modify peptides or proteins is crucial to the production of virtually every biotechnology product • Food processing – the creamy in ice cream • Textile and leather goods – bio-bleaches • Detergents – enzymes to dissolve stains • Paper manufacturing and recycling – reduce negative environmental impacts • Adhesives – barnacles and mussels • Bioremediation – proteins used to clean up harmful waste

5. Proteins as Biotech Products • Therapeutic Protein • used to treat a disease that is caused by a gene that fails to produce a necessary protein or that produces a dysfunctional protein

6. Protein Synthesis in a Eukaryotic Cell. In a eukaryotic cell, DNA is located within chromosomes in the nucleus. The mRNA transcripts carry the DNA code out to the ribosomes, which translate the code into a strand of amino acids.

7. Protein Synthesis: Transcription • Protein synthesis is a two-step process: • First Step - Transcription • Genetic code must be rewritten onto a messenger molecule

8. RNA Structure • Uracil instead of thymine • Ribose sugar instead of deoxyribose sugar • Single stranded • Various shapes • Can leave the nucleus

9. RNA StructureTranscription & Translation requires 3 types of RNA • mRNA – RNA copy of DNA that carries genetic information from the nucleus to the ribosomes • rRNA – makes up the ribosomes • tRNA – carries amino acids to ribosomes for protein synthesis

10. Transcription • RNA polymerase binds to a promoter region on double stranded DNA and unzips the double helix.

11. Transcription • Free RNA nucleotides pair with the complementary DNA of the template strand

12. Transcription mRNA is Processed mRNA leaves nucleus • Introns are spliced out • 7 methyl guanosine cap • Poly-A tail • Travels to the ribosomes in the cytoplasm ribosome nucleus

13. Let’s put it all together • Transcription Animation

14. Your Turn!

15. Protein Synthesis: Translation • Protein synthesis is a two-step process: • Second Step - Translation • mRNA nucleotide code is rendered into a sequence of amino acids

16. Translation • A protein is a string of amino acids held together by peptide bondsand do most of the work in a cell

17. Translation

18. Translation

19. Bacterial Protein Synthesis Animation on protein synthesis

20. Translation • Important Definitions • A codon is composed of 3 RNA nucleotides • Each codon codes for one amino acid • Protein does the work in a cell

21. Translation

22. Translation

23. Translation • Use the Genetic Code to translate the following mRNA strand: • CGAGAAGUC

24. Translation • Always begins at a start codon and ends at a stop codon. • The region between the start and stop codons is called the open reading frame (ORF)

25. Practice • Click on the animation to transcribe and translate a gene. • This is great practice! • HINT! This will be on your exam! Click to see animation

26. Translation Initiation • mRNA attaches to the small subunit of a ribosome • tRNA anticodon pairs with mRNA start codon • Large ribosomal subunit binds and translation is initiated amino acid tRNA anticodon

27. Translation Elongation • Anticodon of tRNA carrying next amino acid binds to codon on mRNA • A peptide bond joins the amino acids and the first tRNA is released.

28. Translation Termination • Amino acid chain continues until a stop codonis read. The amino acid chain is released and all of the translation machinery is recycled to translate another protein.

29. Let’s put it all together • Click on the animation below Translation Animation Translation Video

30. Putting it all together…. Coding: Template: mRNA: tRNA: amino acid: 5’-GATCTGAATCGCTATGGC-3’ 3’-CTAGACTTAGCGATACCG-5’ mRNA 5’-GAUCUGAAUCGCUAUGGC-3’ CUAGACUUAGCGAUACCG Asp, Leu, Asn, Arg, Tyr,Gly

31. Protein Structure • Once the amino acid chain is released from the ribosome, a number of modifications are made in order for the protein to perform it’s intended function. • The protein must fold into it’s appropriate 3-dimensional shape.

32. Protein Structure • Proper folding of the protein is essential for it’s activity because it must bind it’s substrate to perform it’s job.

33. Protein Structure • Primary – Peptide bonds in a chain of amino acids • Secondary – Hydrogen bonding between amino acids forms alpha-helices and beta-sheets • Tertiary – three dimensional folding of protein due to disulfide linkages and hydrophobic interactions between alpha-helices and beta-sheets • Quaternary – aggregation of multiple polypeptide chains

34. Protein Structure • Primary – Peptide bonds in a chain of amino acids

35. Protein Structure • Secondary – Hydrogen bonding between amino acids forms alpha-helices and beta-sheets

36. Protein Structure • Tertiary – three dimensional folding of protein due to disulfide linkages and hydrophobic interactions between alpha-helices and beta-sheets

37. Protein Structure • Quaternary – aggregation of multiple polypeptide chains

38. Protein Structure • Glycosylation • A glycoprotein is a protein on which sugar groups have been added • Increases solubility, orients protein in membrane, extends life of protein • Occurs in the golgi

39. Protein Structure • Another group of proteins, the antibodies is structurally interesting and functionally very important • The function of an antibody is to recognize and bind foreign proteins or other molecules called antigens, for removal • Each type of antibody has the same basic shape

40. Antibodies - Application • Most antibodies are very specific and bind to distinct regions of molecules called epitopes • In the lab, antibodies can be used to bind certain molecules under study, or for diagnosis • A common test used to determine the presence of a protein in solution is the ELISA – Enzyme Linked Immunosorbent Assay

41. Enzymes: Protein Catalysts • Enzymes are proteins that act as catalysts • Enzymes are involved in virtually every reaction in a cell • Many companies have focused on producing enzymes for sale • The molecules upon which enzymes act are called substrates

42. Factors that affect Enzyme Activity • Amount of substrate in a solution • Temperature of a reaction • Acidity or alkalinity

43. Working with Enzymes • Most proteins are kept at cold temperatures to prevent degradation • They are stored in fridges, cold rooms or freezers

44. Studying Proteins SDS-PAGE Animation A technician loads protein samples on a vertical gel. Vertical gel boxes operate in a fashion similar to horizontal gel boxes. Watch this video! How to run SDS-PAGE

45. Studying Proteins Vertical Gel Electrophoresis. Although vertical gel boxes vary from one manufacturer to another, all are basically of the same design. The gel cassettes are snapped or screwed in place (right). Running buffer is added behind the gel, covering the wells. Buffer is poured in the front of the gel cassette to cover the front opening. When the top is placed on the box (left) and the power is turned on, electricity flows from the top (negative charge) to bottom (positive charge). Negatively charged samples move down the gel toward the positive electrode.

46. Studying Proteins • SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) • Separates proteins in an electrical field based on molecular size

47. Studying Proteins • Sodium Dodecyl Sulfate (SDS) • A detergent that denatures the secondary and tertiary structure of the protein • Coats the protein with negative charges Add SDS

48. Studying Proteins • Polyacrylamide Gel Electrophoresis (PAGE) • Much tighter gel matrix than agarose, which makes polyacrylamide ideal for separating proteins

49. Studying Proteins • SDS-PAGE – Proteins separate by size • If run molecular weight marker at the same time, can determine molecular weight of protein • The smaller the peptide chain the faster it moves through the gel

50. Studying Proteins • SDS-PAGE: Gels of different concentration