SURVEY OF BIOCHEMISTRY Amino Acids and Proteins Begin lecture with a 5-min study exercise on naming amino acids! Student with the highest number correct will get a prize.

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?

Amino Acids: Nonpolar Side Chains

Amino Acids: Polar, Uncharged Side Chains

Cysteine & Disulfide Bonds

Cysteine & Disulfide Bonds

Amino Acids: Polar, Uncharged Side Chains

Amino Acids: Polar, Uncharged Side Chains

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

pKa and pI pI = Isoelectric point the pH at which a molecule carries no net electric charge pKa2 pKa1 pI = [pKa1 + pKa2]

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

Peptide Bond Peptide bond links one amino acid to another.

Torsion Angles in Proteins Peptides typically assume the trans conformation

Torsion Angles Defined… Psi: angle made from atoms N-Calpha-Ccarbonyl-Nn+1

Torsion Angles Defined… Phi: angle made from atoms Ccarbonyl,n-1-Nn-Calpha-Ccarbonyl,n

Hydrogen bonding stabilizes the alpha helical structure! 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!

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

Protein Purification Overview Courtesy of

Salting Out: (NH4)2SO4

Gel Filtration Chromatography Separation based on Size

Ion Exchange Chromatography Separation based on Charge

Affinity Chromatography What could be to cause proteins to elute off of an affinity column?

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

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?

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? Must know the value of 0.75 Conc. = (43,824 M-1cm-1) x (1 cm) Conc. = 0.00001711 M = 17 µM

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

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 proportional to the protein concentration. ENDED HERE… How is data generated? How is data analyzed?

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 concentration in the same manner as the standards and compare - visually and spectrophotometrically!

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.