2. Intended Learning Objectives You should be able to… 1. Give 3 examples of proteins that are important to humans and are currently produced by transgenic organisms. 2.Describe a protein’s composition. 3.Recognize the 4 basic protein structures. 4.Explain the following key terms: Hydrophobic and Hydrophilic. 5.Honors: Identify the parts of an amino acid. Resources: Chapter 3 (3.14) and Notes

3. Intended Learning Objectives You should be able to… 6. Explain how we will be able to isolate the GFP protein for purification. Resources: Lab Handouts and Notes

4. Intended Learning Objectives You should be able to… 6a. Explain what each of the following do in order to help extract the GFP. - Lysozyme - Freezer - Centrifuge (1 st and 2 nd uses)

5. Intended Learning Objectives You should be able to… 6b. Explain what each buffer did to the proteins in the column. Be sure to indicate the concentration of salt in each. - Equilibration buffer - Binding buffer - Wash buffer - Elution buffer

8. Hydrophobic Interactions Supernatant Low salt buffer

9. Protein Structure 1 o Primary 2 o Secondary 3 o Tertiary 4 o Quaternary

11. 1 ° Primary Structure Linear sequence of amino acids that make up the polypeptide chain. Determined by the genetic code in particular the mRNA The bond between two amino acids is a peptide bond. This bond is formed by the removal of a H 2 O molecule from two different amino acids, forming a dipeptide. The sequence of amino acids determines the positioning of the different chemical groups of each building block which determines which groups will interact with each other and therefore determines the final structure and function of the molecule.

12. 2 ° The secondary structure coiling of the peptide chain into a helix or some other regular pattern of twists or kinks of the polypeptide chain. due to hydrogen bonds forming between the atoms of the amino acid backbone of the polypeptide chain. The two most common types of secondary structure are called the alpha helix and beta pleated sheet.

13. 3 ° Tertiary structure three dimensional globular structure formed by bending and twisting of the polypeptide chain. folding of the polypeptide chain is stabilized by multiple weak, non-covalent interactions. These interactions include: + Hydrogen bonds - form when a Hydrogen atom is shared by two other atoms. + Electrostatic interactions - occur between charged amino acid side chains. + Hydrophobic interactions - amino acids with a polar (water soluble) side chain are often found on the surface of the molecule while amino acids with nonpolar (water insoluble) side chain are buried in the interior. This means that the folded protein is soluble in water or aqueous solutions. + Disulfide Bonds: The polypeptide chains of some proteins are linked by disulfide bonds.

14. Four levels of Organization of Protein Structure 1 ° Primary Structure refers to the linear sequence of amino acids that make up the polypeptide chain. This sequence is determined by the genetic code, the sequence of nucleotide bases in the DNA. The bond between two amino acids is a peptide bond. This bond is formed by the removal of a H 2 O molecule from two different amino acids, forming a dipeptide. The sequence of amino acids determines the positioning of the different chemical groups of each building block which determines which groups will interact with each other and therefore determines the final structure and function of the molecule. 4 ° Quaternary structure. Proteins that contain more than one polypeptide chain Each polypeptide chain in the protein is called a subunit. The subunits can be the same polypeptide chain or different ones. For some proteins, quaternary structure is required for full activity (function) of the protein.

23. Hydrophobic interaction chromatography H I C The background – hydrophobic compounds stick together – association affected by: salt concentration  greater concentration = more hydrophobic interactions The idea – stick GFP to a polymer sphere with hydrophobic groups – wash it off by changing the salt concentration ( decrease or increase?) Hydrophobic region

24. True or False 1.The function of a protein is determined by its structure/shape. 2.Hydrophobic molecules tend to “hang out” or bind to other hydrophobic molecules. 3.Higher concentrations of salt decrease the binding of proteins to hydrophobic polymers. 4.Molecules that are Hydrophobic are polar molecules. 5.Electrostatic attractions between amino acids occur in the primary structure of a protein. TRUE FALSE TRUE FALSE

25. Which of the following is the strongest bond? Polar covalent Nonpolar covalent Hydrogen Electrostatic

26. Which protein structure does the picture depict below? a-helix or beta sheet? Primary, secondary, tertiary, or quaternary structure? a-helix secondary

27. Which of the following weakened the cell wall of the bacteria cells. lysozyme freezer centrifuge arabinose

28. Which of the following had the highest salt concentration? Wash buffer Equilibration buffer Elution Buffer Binding Buffer

29. Biopolymer (phenyl agarose - Binding Surface) Driving force for hydrophobic adsorption Water molecules surround the analyte and the binding surface. When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy. Temperature plays a strong role Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer Hydrophobic Interaction Chromatography Hydrophobic region (Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

30. Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobicity. Molecules that contain both hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer. The salt in the buffer reduces the solvation of the sample. As overall solvation decreases, hydrophobic regions that become exposed are adsorbed by the medium. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity.