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Review: Amino Acid Side Chains

R. a. a. vs. H. N. N. 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. . R. H. f. C a. C a. C a. H. R. H.

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Review: Amino Acid Side Chains

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  1. R a a vs H N N 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

  2. R H f Ca Ca Ca H R H R Review: Backbone Conformation • Side chains collision also limit f/ combinations • Backbone restricted  Secondary structure limited

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

  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

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