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:Polar, Charged Be able to draw,name and give1-letter/3-lettercodes for all 20amino acids!
pI = [pKa1 + pKa2] pKa and pI • pI = Isoelectric pointthe pH at which a molecule carries no net electric charge pKa2 pKa1
Polypeptide SequenceNomenclature Be able to drawa polypeptide! Tetrapeptide: AYDG
Peptide Bond Peptide bond links one amino acid to another.
Torsion Angles in Proteins Peptides typically assumethe trans conformation
Torsion Angles Defined… • Psi: angle made from atomsN-Calpha-Ccarbonyl-Nn+1
Torsion Angles Defined… • Phi: angle made from atomsCcarbonyl,n-1-Nn-Calpha-Ccarbonyl,n
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!
Beta Sheets Notice the hydrogen bondingfrom the carbonyloxygen to the amino hydrogen
Courtesy of Protein Purification Overview
Gel Filtration Chromatography Separation based on Size
Ion Exchange Chromatography Separation based on Charge
Affinity Chromatography Affinity What could be to cause proteinsto elute off of anaffinity 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?
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
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
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?
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!
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.