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