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SURVEY OF BIOCHEMISTRY Amino Acids and Proteins

SURVEY OF BIOCHEMISTRY Amino Acids and Proteins

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SURVEY OF BIOCHEMISTRY Amino Acids and Proteins

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  1. SURVEY OF BIOCHEMISTRYAmino Acids and Proteins

  2. Amino Acids Can you give the 1-letter and 3-letter names for all 20 amino acids within 5 minutes? Can you draw a oligopeptide of any given sequence? With correct stereochemistry?

  3. Amino Acids: Nonpolar Side Chains

  4. Amino Acids: Polar, Uncharged Side Chains

  5. Cysteine & Disulfide Bonds

  6. Cysteine & Disulfide Bonds

  7. Amino Acids: Polar, Uncharged Side Chains

  8. Amino Acids: Polar, Uncharged Side Chains

  9. Amino Acids:Polar, Charged Be able to draw,name and give1-letter/3-lettercodes for all 20amino acids!

  10. pI = [pKa1 + pKa2] pKa and pI • pI = Isoelectric pointthe pH at which a molecule carries no net electric charge pKa2 pKa1

  11. Polypeptide SequenceNomenclature Be able to drawa polypeptide! Tetrapeptide: AYDG

  12. Peptide Bond Peptide bond links one amino acid to another.

  13. Torsion Angles in Proteins Peptides typically assumethe trans conformation

  14. Torsion Angles Defined… • Psi: angle made from atomsN-Calpha-Ccarbonyl-Nn+1

  15. Torsion Angles Defined… • Phi: angle made from atomsCcarbonyl,n-1-Nn-Calpha-Ccarbonyl,n

  16. Alpha Helix Sequence coils in a right-handedmanner.Notice hydrogen bonding along the helical axis from carbonyl oxygen(of residue n) to the amino hydrogenof residue (n+4). Hydrogen bonding stabilizes the alpha helical structure!

  17. Beta Sheets Notice the hydrogen bondingfrom the carbonyloxygen to the amino hydrogen

  18. Courtesy of Protein Purification Overview

  19. Salting Out: (NH4)2SO4

  20. Gel Filtration Chromatography Separation based on Size

  21. Ion Exchange Chromatography Separation based on Charge

  22. Affinity Chromatography Affinity What could be to cause proteinsto elute off of anaffinity column?

  23. Ways to Assess Protein Purification • Total Protein Concentration Assays • Beer’s Law • Colorimetric Assays • Specific Protein Assays • Activity Assays • Immunoassays • ELISA • RIA

  24. Beer’s Law What is the molar concentration of a solution of Bovine Serum Albumin (BSA) that exhibits an A280 of 0.75 with a path length of 1 cm?

  25. Must know the value of Beer’s Law What is the molar concentration of a solution of Bovine Serum Albumin (BSA) that exhibits an A280 of 0.75 with a path length of 1 cm? 0.75 Conc. = (43,824 M-1cm-1) x (1 cm) Conc. = 0.00001711 M = 17 µM

  26. Bradford Protein Assay Suppose you have a protein mixture and you need to determine the protein concentration. You cannot use Beer’s Law.Because you would not know the extinction coefficientfor the protein mixture at 280 nm Alternative approach: Bradford Protein Assay

  27. Bradford Protein Assays Bradford Protein Assay reagent contains Coomassie brilliant blue which reacts with basic (esp. Arg) and aromatic amino acids to yield a blue color with intensity proportionalto the protein concentration. How is data generated? How is data analyzed?

  28. Data Generation: Bradford Assay Reference contains only buffer Create a series of protein standards at known increasing concentrations and mix with the Bradford reagent 2.5 5 10 15 20 25 µg/mL Treat the protein mixture of unknown concentrationin the same manner as the standards and compare - visually and spectrophotometrically!

  29. Bradford Assay Data Analysis Graph the absorbance at 595 nm as a function of [standard] in µg/mL. Fit the line and use itto deduce the [protein] in the mixture based onits absorbance.