1 SURVEY OF BIOCHEMISTRY Proteins and Biomolecular Stability

2 Protein Structure Primary (1°): amino acid sequence Secondary (2°) –Alpha Helix –Beta Sheet Tertiary (3°) Quaternary (4°)

3 Primary and Secondary Structure Myoglobin - 2V1K Superoxide Dismutase - 1XSO

4 Recap Structures of 20 amino acids pK a and pI 1°: Polypeptide Sequence 2°: Secondary Structures –Alpha Helices –Beta Sheets

5 Recap continued Protein Purification Methods –Gel Filtration –Ion Exchange –Affinity How to assess purification? –Purity –Yield

6 SDS-PAGE Electrophoresis: a method for separating molecules based on size and charge when exposed to an electric field. Name “SDS-PAGE”: SDS = sodium dodecyl sulfate PAGE = polyacrylamide gel electrophoresis

7 Sodium Dodecyl Sulfate SDS confers negative charge on proteins and denatures proteins Sodium Dodecyl Sulfate (Lauryl Sulfate) CH 3 (CH 2 ) 11 OSO 3 Amphiphilic Hydrophilic Hydrophobic Anionic Detergent in a wide variety of products Proteins are primarily denatured by boiling them prior to electrophoresis!

8 Buffers maintain pH control Allow gel to fully polymerize Stacking Gel 0.5M Tris-HCl pH 6.8 Resolving Gel 1.5M Tris-HCl pH 8.8 SDS-PAGE Buffers

9 Ensure that sample has fully dentured! Stacking Gel 0.5M Tris-HCl pH 6.8 Resolving Gel 1.5M Tris-HCl pH 8.8 In the GelIn the Sample Laemmli Sample Buffer 0.5M Tris-HCl,pH 6.8 SDS Glycerol Bromophenol Blue Sample Preparation Boil Sample for 1-5 min

10 Electrophoresis Buffer H 3 N-CH 2 -COOH + Acidic Form H 3 N-CH 2 -COO + - Zwitterionic Form Basic Form H 2 N-CH 2 -COO - Fully Protonated Loss of 1 Proton Loss of 2 Protons Tris Base, Glycine, SDS Electrophoresis Buffer

11 Stacking Gel 0.5M Tris-HCl pH 6.8 Resolving Gel 1.5M Tris-HCl pH 8.8 In the Gel H 3 N-CH 2 -COO H 2 N-CH 2 -COO + - Zwitterion Form - Basic Form SDS-PAGE Gly lags Gly leads Note: Discontinuous SDS-PAGE is depicted here!

Migration in an SDS-PAGE Gel

13 Migration in an SDS-PAGE Gel Stop electrophoresis when dye front reaches bottom of gel Stain with Coomassie

14 Purity Purity is a measure of how undefiled a protein sample is. Lots of impurities Pure protein

15 Yield % Yield = Amount of protein recovered Amount of protein initially Example: % Yield = (208 / 358.2) x 100 = 58.1% After 2 steps of purification

16 Protein Sequencing Separate subunits Dansyl Chloride Reaction Proteolytic Digestion Cyanogen Bromide Cleavage Edman Degradation Read on your own! Study how each works!

17 How to separate subunits? Dithiothreitol, DTT2-Mercaptoethanol

18 Proteolytic Digestion Know this!

19 Proteolytic Digestion How many fragments would result from digestion with trypsin?

20 Proteolytic Digestion Trypsin cleaves after Lys (K) and Arg (R): 16 fragments!

21 Protein Structure Classifications 1°: amino acid sequence 2°: local spatial arrangement of a polypeptide backbone without regard for side chains 3°: 3D structure of a protein including its side chains 4°: spatial arrangements of subunits

22 Tertiary Folds Alpha Beta Some proteins only have alpha helices (plus turns and random coils). Others only have beta sheets (plus turns and random coils).

23 Alpha/Beta Tertiary Folds Some proteins have a combination of alpha helices and beta sheets (plus turns and random coils).

24 Biomolecular Stability Nucleic acids and proteins are stabilized by the same types of intermolecular forces. Hydrophobic Effect: the tendency of water to minimize its contact with hydrophobic groups in molecules. How does the hydrophobic effect impact proteins and nucleic acids?

25 Entropy Entropy measures the spontaneous dispersal of energy: how much energy is spread out in a process -or- how widely spread out it becomes — at a specific temperature

26 Entropy & the Hydrophobic Effect How is entropy increased by the hydrophobic effect?

27 How do bond energies compare? Type of BondBond Strength (kJ/mol) Covalent Ionic Interaction86 Hydrogen Bond20 Dipole-dipole9.3 London Dispersion0.3 Table 2-1 Relatively speaking, H-bonds are weak, but they are not nearly as weak as one might expect!

28 Why Do Base Pairs Stack? ENTROPY Hydrophobic effect induces release of water “binding” to DNA bp’s such that the hydrophobic ring systems can stack on top of each other to minimize contact with water. Consider the magnitude of stacking energy… Etc.

29 Forces Stabilizing Biomolecule Structure ProteinsNucleic Acids Hydrophobic EffectHydrophobic Effect Globular shape Base Stacking Disulfide Bonds H-BondsH-bonds Alpha Helices Base Pairing Beta Sheets Ionic InteractionsIonic Interactions Salt Bridges Metal Ions