Review: Amino Acid Side Chains

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
2
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 (gt50),
  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