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Proteins

Proteins. Biotech 2. Arabinose binding protein from E. coli. What Are Proteins?. Made of a polymer of amino acids (a.a.) Responsible for: Catalyzing reactions (enzymes) Regulating gene expression (regulatory proteins) Building cells, tissues, viruses etc. (structural proteins)

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Proteins

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  1. Proteins Biotech 2 Arabinose binding protein from E. coli

  2. What Are Proteins? • Made of a polymer of amino acids (a.a.) • Responsible for: • Catalyzing reactions (enzymes) • Regulating gene expression (regulatory proteins) • Building cells, tissues, viruses etc. (structural proteins) • Transporting materials into and out of a cell, responsible for muscle contraction, motion of flagella etc. • Remember: there are 20 a.a. (the order of them determines the specific protein) • Usually 100 to 1000 a.a. long- Measured in Daltons (Da) (each amino acid is approx. 110 Da)

  3. Abundance of Protein • Proteins are the most abundant molecules present in all living cells • More than half of an organisms cellular dry weight is protein • Typical mammalian cells have at least 10,000 different types of proteins

  4. Amino Acids Review • Every a.a. has a carbon (the alpha carbon) • Attached to the alpha carbon is: • A carboxyl group (-COOH) • An amino group (-NH2) • A side chain (R group) Carboxyl group Amino group

  5. Dehydration Reactions • Amino acids join together via a dehydration reaction • A peptide bond forms between them • Amino Acids always build onto the CARBOXYL END or (C terminal end)

  6. Four Groups of Amino Acids(based on the characteristics of the R groups) • Non-polar (hydrophobic) • Polar (uncharged, hydrophilic) • Negatively charged (acidic) • Positively charged (basic)

  7. pH Review(potential for Hydrogen atoms to form) • pH: measure of the amount of hydrogen ions [H+]in a solution • Ionization: the separation of molecules into positive and negative ions • Ex. NaCl  Na+ + Cl- • Acids: releases H+ ions in solution • Bases: accept H+ ions

  8. pH Review cont.-The pH Scale- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Acidic Neutral Basic (high H+ concentration) (low H+ concentration)

  9. The pH Scale cont. • It is logarithmic: • A solution with a pH of 5 is 10 times more acidic than a solution with a pH of 6 (it has 10 times the concentration of H+ ions) • A solution with a pH of 4 is 100 times more acidic than one with a pH of 6

  10. Buffers • Weak acids and weak bases can ionize and re-associate in water • These compounds play an important role in living systems-they act as buffers • Buffers minimize changes in pH by donating or accepting H+ or OH- ions

  11. Isoelectric Point (pI) At a certain pH, a protein will contain an equal number of positive and negative charges making it electrically neutral, the pH at which this occurs is called the Isoelectric Point or pI

  12. Protein Folding • To be biologically active, proteins must fold • Most proteins are highly folded • The way it’s folded depends on the a.a. order • The a.a. tend to fold so that the charged a.a. are on the outside to interact with other proteins, elements, etc. • Folding: • Primary structure • Secondary structure • Tertiary structure • Quaternary structure • Native structure

  13. Primary Structure • The newly formed chain of amino acids • Linear met-his-pro-tyr-lys-lys-his-pro-pro-try-asp-asp-pro N-terminus C-terminus

  14. Secondary Structure • The linear amino acid sequence begins to fold into: • Alpha helices • Beta pleated sheets • Secured by hydrogen bonds between the amino acids

  15. Alpha Helix Alpha helix

  16. Beta Sheets • Beta -sheets are composed of 2 or moredifferent regions of amino acids Beta sheet

  17. Tertiary Structure • Tertiary structure refers to the complete three-dimensional structure of the polypeptide units of a given protein

  18. Quaternary Structure • More than one protein interacting

  19. Hemoglobin-an example of a quaternary structure- • Hemoglobin • Made of 4 proteins (it’s tetrameric or a tetramer) • Trimeric (made of 3 proteins) • Dimeric (made of 2 proteins) • Monomeric (made of 1 protein) • Multimeric (made of >1 protein)

  20. Structural/Functional Domains • Proteins have areas that form specific functions: called Functional Domains • Proteins have areas that offer support: called Structural Domains

  21. Protein Modificatoins Some proteins are modified with other functional groups, such as Phosphorylation and Glycosylation • Phosphorylation (addition of phosphate) can often control the activity of a protein • Glycosylation(addition of sugars) which can aid in the function of the protein and direct where the protein will go .

  22. Protein Purification • The characteristics of the amino acids that make up the protein will determine the type of chromatographyused to purify the protein Liquid Chromatography

  23. Separation of Proteins-Chromatography- • Inexpensive, easy, and efficient • Separate proteins based on: • Size • Size exclusion chromatography (gel permeation or gel filtration chromatography) • Charge • Ion-exchange chromatography • Hydrophobicity • Hydrophobic interaction chromatography, reverse phase chromatography • Function • Affinity Chromatography

  24. Size Exclusion chromatography(separation based on size of the proteins) • Protein size is measured as molecular weight (MW) in daltons (d). • A dalton is the approximate mass of a hydrogen atom • Sizes of proteins range from a few thousand to millions of daltons. • The column matrix consists of small circular beads (molecular sieves) • The beads are cross-linked using carbohydrates and pores are created • The size of the pores is determined by the amount of cross-linking. • When a mixture of proteins is applied to the column: • Small proteins will enter the pores and move slowly down the column • Large proteins won’t be able to enter the pores and will pass quickly down the column

  25. Ion-Exchange Chromatography(separation based on charge of the proteins) • Relies on differences in the acidic and basic properties of the proteins • The overall charge of the protein can be altered by changing the pH • If the pH of the protein is raised above the pI, it will become negatively charged • If the pH of the protein is dropped below the pI, it will become positively charged • The matrix is made of small semi-solid spheres called resins

  26. Ion-Exchange Chromatography Cont.(separation based on charge of the proteins) • The resins contain ionizable groups • A resin is a cation exchanger when the matrix is acidic (interacts with positively charged proteins) • A resin is an anion exchanger when the matrix is basic (interacts with negatively charged proteins) • Changing the pH of the matrix will control the movement of protein through it!

  27. Hydrophobic Interaction Chromatography(separation based on hydrophobicity of the proteins) • Relies on the hydrophobic residues of the proteins • Speed through the column depends on the hydrophobicity of the protein

  28. Affinity Chromatography(separation based on function of the proteins) • Relies on binding interactions between antigens and antibodies or substrates and enzymes • Example: • Matrix contains antibody specific to the protein of interest • Protein of interest will be retained; all other molecules will flow through

  29. Using a Spectrophotometer to Analyze Proteins • Peptide bonds in proteins absorb UV light • You can use this characteristic to quantify the amount of protein you have

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