1. Review: Amino Acid Side Chains Aliphatic- Ala, Val, Leu, Ile, Gly Polar- Ser, Thr, Cys, Met, [Tyr, Trp] Acidic (and conjugate amide)- Asp, Asn, Glu, Gln Basic- Lys, Arg, His Aromatic- Phe, Tyr, Trp, [His] Proline N  N  H R vs

2. Review:  ackbone Conformation  Side chains collision also limit  /  combinations  Backbone restricted  Secondary structure limited CC HR   CC HR CC HR

3. Review: Heirarchy of Structure Primary- sequence Secondary- local Supersecondary (motifs)- intermediate Domains- independent folding units Tertiary- organization of a complete chain Quaternary- organization of multiple chains

4. Review: Tertiary Structure Soluble proteins have an inside (core) and outside  Folding driven by water- hydrophilic/phobic  Side chain properties specify core/exterior   Some interactions inside, others outside Specific structures result from side chain interactions  Hydrophobic interactions (interior)  Hydrogen bonds (interior and exterior)  Ionic Interactions (exterior)

5. Relationships Among Proteins Many sequences can give same tertiary structure  Side chain pattern more important than sequence When sequence homology is high (>50%), probably same structure and function (structural genomics)  Cores conserved  Surfaces and loops more variable *3-D shape more conserved than sequence* *There are a limited number of structural frameworks*

6. Relationships Among Proteins I. Homologous: conserved sequence (cytochrome c)  Same structure  Same function  Modeling structure from homology II. Similar function- different sequence (dehydrogenases)  One domain same structure  One domain different III. Similar structure- different function (cf. thioredoxin)  Same 3-D structure  Not same function

7. How to Tell Proteins Apart! Sequence and fold give overall properties  Molecular weight  Solubility  Exposed hydrophobic surface  Ability to bind other molecules, metals  pI- the overall charge of the protein  Sequence!!! *To characterize properties, separate the protein from all other cell contents*

8. Protein Purification Techniques A. Simple solubility characteristics- precipitation  Temperature  pH  “Salting out” *Different proteins precipitate under different solution conditions- can use soluble or insoluble fractions*

9. Protein Purification Techniques B. Chromatography - fractionation of contents in solution based on selection by a stationary phase  Size- sieve effect, small molecules faster  Ion exchange- charge attraction at protein surface  Choose “+” stationary phase for proteins with more “-” charge  First bind everything, then elute with salt  Hydrophobic interaction- hydrophobic accessible surface  Affinity chromatography  Antibody, binding protein  Inserted tag (e.g. 6-His)

10. Protein Purification Techniques C. Gel Electrophoresis- migration in a gel matrix (size and shape) driven by an electric field (charge)  Sieving effect  Relative charge  Visualization- staining with dye, fluorescent antibody (Western blotting)  SDS- protein denaturant, enables separation based almost exclusively on molecular weight  Iso-electric focusing- method to measure pI, but also can be used for separation

11. I Volume (ml) (Lanes 1, 2) (Lanes 3, 4) (Lanes 7, 8, 9) M 1 2 3 4 5 6 7 8 9 T-ag GST Fusion protein 14.4 21.5 36.5 54.4 Chromatography and SDS-PAGE

12. Protein Characterization A. Sequence  Amino acid analysis- total digest, then count how much of each amino acid  Edman stepwise degradation- cleave of one residue at a time, then identify  Peptide mapping- cleave into fragments, then identify  Direct sequencing by Mass Spectrometry  Exact molecular weights  Characteristic fragmentation

13. Protein Characterization B. Spectroscopic properties  UV-Vis- Backbone, Phe, Tyr, Trp, co-factors  Infrared/Raman- characteristic bond vibrations  Circular Dichroism (CD)- backbone conformation  Fluorescence  Intrinsic- Trp, Tyr  Attached dyes- Cys  Electron Paramagnetic Resonance (EPR)  Metals, free radicals  Attached probes  Nuclear Magnetic Resonance (NMR)  Many probes viewed simultaneously  Structure and dynamic processes

14. Protein Characterization C. Antibodies  Use protein of interest to raise antibodies (rabbit)  Different antibodies can recognize different regions (epitopes)  Can distinguish differences as small as 1 residue  Attachment of indicators- dyes, radioactivity  Applications- e.g. immunoassay, ELISA