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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. - PowerPoint PPT Presentation
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Review: Amino Acid Side ChainsReview: 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
Review: Review: ackbone Conformationackbone Conformation
Side chains collision also limit /combinations
Backbone restricted Secondary structure limited
C
HR
C
H R
C
H R
Review: Heirarchy of StructureReview: 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
Review: Tertiary StructureReview: 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)
Relationships Among ProteinsRelationships 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*
Relationships Among ProteinsRelationships Among ProteinsI. 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
How to Tell Proteins Apart!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*
Protein Purification TechniquesProtein Purification Techniques
A. Simple solubility characteristics- precipitation
Temperature
pH
“Salting out”
*Different proteins precipitate under different solution conditions- can use soluble or insoluble fractions*
Protein Purification TechniquesProtein Purification Techniques B. Chromatography- fractionation of contents in
solution based on selection by a stationary phase1. Size- sieve effect, small molecules faster
2. Ion exchange- charge attraction at protein surfaceChoose “+” stationary phase for proteins with
more “-” chargeFirst bind everything, then elute with salt
3. Hydrophobic interaction- hydrophobic accessible surface
4. Affinity chromatographyAntibody, binding protein Inserted tag (e.g. 6-His)
Protein Purification TechniquesProtein 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
I
Volume (ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
0 20 40 60 80 100 120 140
(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
14.421.5
36.5
54.4
Chromatography and SDS-PAGE
Protein CharacterizationProtein Characterization A. Sequence
1. Amino acid analysis- total digest, then count how much of each amino acid
2. Edman stepwise degradation- cleave of one residue at a time, then identify
3. Peptide mapping- cleave into fragments, then identify
4. Direct sequencing by Mass Spectrometry
Exact molecular weights
Characteristic fragmentation
Protein CharacterizationProtein Characterization B. Spectroscopic properties
1. UV-Vis- Backbone, Phe, Tyr, Trp, co-factors2. Infrared/Raman- characteristic bond vibrations3. Circular Dichroism (CD)- backbone conformation4. Fluorescence
Intrinsic- Trp, TyrAttached dyes- Cys
5. Electron Paramagnetic Resonance (EPR)Metals, free radicalsAttached probes
6. Nuclear Magnetic Resonance (NMR)Many probes viewed simultaneouslyStructure and dynamic processes
Protein CharacterizationProtein 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