1 SURVEY OF BIOCHEMISTRY Amino 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

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7 Amino Acids: Polar, Uncharged Side Chains

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9 Amino Acids: Polar, Charged Be able to draw, name and give 1-letter/3-letter codes for all 20 amino acids!

10 pK a and pI pI = Isoelectric point the pH at which a molecule carries no net electric charge pK a1 pK a2 pI = [pK a1 + pK a2 ]

11 Polypeptide Sequence Nomenclature Tetrapeptide: AYDG Be able to draw a polypeptide!

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

13 Torsion Angles in Proteins Peptides typically assume the trans conformation

14 Torsion Angles Defined… Psi: angle made from atoms N-C alpha -C carbonyl -N n+1

15 Torsion Angles Defined… Phi: angle made from atoms C carbonyl,n-1 -N n -C alpha -C carbonyl,n

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

17 Beta Sheets Notice the hydrogen bonding from the carbonyl oxygen to the amino hydrogen

18 Protein Purification Overview Courtesy of

19 Salting Out: (NH 4 ) 2 SO 4

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 proteins to elute off of an affinity 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 A 280 of 0.75 with a path length of 1 cm?

25 Beer’s Law What is the molar concentration of a solution of Bovine Serum Albumin (BSA) that exhibits an A 280 of 0.75 with a path length of 1 cm? Conc. = 0.75 (43,824 M -1 cm -1 ) x (1 cm) Conc. = M = 17 µM Must know the value of

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 coefficient for the protein mixture at 280 nm Alternative approach: Bradford Protein Assay

27 Bradford Protein Assays How is data generated? How is data analyzed? 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 proportional to the protein concentration.

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 µg/mL Treat the protein mixture of unknown concentration in 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 it to deduce the [protein] in the mixture based on its absorbance.