Introduction to Studying Proteins

1. Introduction to Studying Proteins Chapter 5

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.

3. 5.1 The Structure and Function of Proteins • Virtually all biotechnology products have something to do with proteins. • Protein has a three-dimensional structure.

4. Protein Molecule Structure • Polymers composed of amino acids • Twenty different kinds of amino acids are found in protein • Most proteins contain tens of hundreds of amino acids Function of Structural Proteins For an HIV particle to recognize, attach, and infect a T-helper cell, the gp210 structure must be a precise shape and must exactly match its human cell membrane receptors Function of Antibody Proteins Recognize and bind foreign proteins or other molecules (antigens) for removal from the body

5. Vocabulary • X-ray crystallography – a technique used to determine the three-dimensional structure of a protein • Polar – the chemical characteristic of containing both a positive and negative charge on opposite sides of a molecule • Primary structure – the order and type of amino acids found in a polypeptide chain • Secondary structure – the structure of a protein (alpha helix and beta sheets) that results from hydrogen bonding • Tertiary structure – the structure of a protein that results from several interactions, the presence of charged or uncharged “R” groups, and hydrogen bonding • Quaternary structure – the structure of a protein resulting from the association of two or more polypeptide chains • Glycosylated – descriptive of molecules to which sugar groups have been added • CD4 cells – referring to human white blood cells, which contain the cell surface recognition protein CD4 • Reverse transcriptase – an enzyme that transcribes a complementary strand of DNA from a strand of RNA • Antigens – foreign proteins or molecules that are the target of binding by antibodies • Epitope – the specific region on a molecule that an antibody binds to • ELISA – short for enzyme-linked immunospecific assay, a technique that measures the amount of protein or antibody in a solution • Monoclonal antibody – a type of antibody that is directed against a single epitope • Hybridoma – a hybrid cell used to generate monoclonal antibodies that results from the fusion of immortal tumor cells with specific antibody-producing white blood cells (B-cells)

6. 5.1 Review Questions • How many different kinds of amino acids are found in proteins? What distinguishes one amino acid from another? • What causes polypeptide chains to fold into functional proteins? • How many polypeptide chains are found in an antibody, and how are they held together in a protein? • What is the value of monoclonal antibody technology?

7. 5.2 The Production of Protein Until recently, proteins could only be made in cells. Now small polypeptide chains can be synthesized in the laboratory. Overview of Protein Synthesis Protein synthesis occurs continuously throughout a cell’s life Eukaryote and prokaryote – protein synthesis is similar

8. 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.

9. Transcription and Translation Protein synthesis is a two-step process: First Step: Genetic code must be rewritten onto a messenger molecule

10. Second Step: mRNA nucleotide code is rendered into a sequence of amino acids

11. The Importance of Proteins in Biotech R&D The ability to synthesize and modify peptides or proteins is crucial to the production of virtually every biotechnology product.

12. Vocabulary • Protein synthesis – the generation of new proteins from amino acid subunits; in the cell, it includes transcription and translation • Transcription – the process of deciphering a DNA nucleotide code and converting it into RNA nucleotide code; the RNA carries the genetic message to a ribosome for translation into a protein code • Codon – a set of three nucleotides on a strand of mRNA that codes for a particular amino acid in a protein chain • Translation – the process of reading an mRNA nucleotide code and converting it into a sequence of amino acids • tRNA – a type of ribonucleic acid (RNA) that shuttles amino acids into the ribosome for protein synthesis • Peptidyl transferase – an enzyme found in the ribosome that builds polypeptide chains by connecting amino acids into long chains through peptide bonds • Phosphorylation – adding phosphate groups • Cleavage – process of splitting the polypeptide into two or more strands • Taq polymerase – a DNA synthesis enzyme that can withstand the high temperatures used in PCR

13. 5.2 Review Questions • Distinguish between transcription and translation. • If a structural gene’s code is “TAC GGC ATG CCC TTA CGC ATC,” what will the mRNA transcript be? • If the mRNA transcript from question No. 2 were translated into a peptide, what would the amino-acid sequence of the peptide be? • What is the name of the machine that can make small sections of amino-acid chains?

14. 5.3 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 Enzymes and Their Substrates The molecules upon which enzymes act are called substrates Factors That Affect Enzyme Activity Amount of substrate in a solution Temperature of a reaction Acidity or alkalinity

16. Vocabulary • Substrate – the molecule that an enzyme acts on • Cofactors – an atom or molecule that an enzyme requires to function • Lock and key model – a model used to describe how enzymes function, in which the enzyme and substrate make an extract molecular fit at the active site, triggering catalysis • Induced fit model – a model used to describe how enzymes function, in which a substrate squeezes into an active site and induces the enzyme’s activity • Optimum temperature – the temperature at which an enzyme achieves maximum activity • Denaturation – the process in which proteins lose their conformation or three-dimensional shape • Optimum pH – the pH at which an enzyme achieves maximum activity

17. 5.3 Review Questions • Name three examples of enzymes and their substrates. • What happens if an enzyme is at a temperature significantly above its optimum temperature? • What happens if an enzyme is at a pH significantly above or below its optimum level? • What would an enzyme be called if it moved methyl groups (-CH3) between molecules?

18. 5.4 Studying Proteins A technician loads protein samples on a vertical gel. Vertical gel boxes operate in a fashion similar to horizontal gel boxes.

19. 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.

20. Silver stain is much more sensitive than Coomassie® Blue. When samples have low concentrations of protein or DNA, silver-staining is the method of choice.

21. Vocabulary • PAGE – short for polyacrylamide gel electrophoresis, a process in which proteins and small DNA molecules are separated by electrophoresis on vertical gels made of the synthetic polymer, polyacrylamide • Coomassie® Blue – a dye that stains proteins blue and allows them to be visualized • Silver stain – a stain used for visualizing proteins

22. 5.4 Review Questions • What does “PAGE” stand for, and what samples are studied using PAGE? • What separates molecules on a PAGE gel? • PAGE gels are usually run at what amount of current? • A technician has a stock protein solution with a concentration of 1 mg/mL. He prepares a 1:4 serial dilution of the stock and runs the samples on a PAGE gel. What is the preferred method of staining and why?

23. 5.5 Applications of Protein Analysis Protein profile of cells and tissues A protein’s structure can help explain its function Chemical processes in cells Evolution and taxonomic relationships

24. Vocabulary • Taxonomic relationships – how species are related to one another in terms of evolution • Biomanufacturing – industry focusing on the production of proteins and other products created by biotechnology

25. 5.5 Review Questions • What causes the difference between normal and sickled cells in sickle cell disease? • Give an example of proteins studied to understand evolutionary relationships. • What is NCBI, how can you access it, and what important information is found there? • Do all protein scientists work at biotechnology companies? Explain.

26. Questions and Comments?