SURVEY OF BIOCHEMISTRY Proteins and Biomolecular Stability

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SURVEY OF BIOCHEMISTRYProteins and
Biomolecular Stability
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Protein Structure

• Primary (1) amino acid sequence
• Secondary (2)
• Alpha Helix
• Beta Sheet
• Tertiary (3)
• Quaternary (4)

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Primary and Secondary Structure
Superoxide Dismutase - 1XSO
Myoglobin - 2V1K
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Recap

• Structures of 20 amino acids
• pKa and pI
• 1 Polypeptide Sequence
• 2 Secondary Structures
• Alpha Helices
• Beta Sheets

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Recap continued

• Protein Purification Methods
• Gel Filtration
• Ion Exchange
• Affinity
• How to assess purification?
• Purity
• Yield

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SDS-PAGE

• Electrophoresis a method for separating
  molecules based on size and charge when exposed
  to an electric field.

Name SDS-PAGE SDS sodium dodecyl
sulfate PAGE polyacrylamide gel
electrophoresis
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Sodium Dodecyl Sulfate
SDS confers negative charge on proteins and
denatures proteins
Amphiphilic
Anionic Detergent in a wide variety of products
Hydrophilic
Hydrophobic
Sodium Dodecyl Sulfate(Lauryl Sulfate)
Proteins are primarily denatured by boiling them
prior to electrophoresis!
CH3(CH2)11OSO3
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SDS-PAGE Buffers
Buffers maintain pH control Allow gel to fully
polymerize
Stacking Gel 0.5M Tris-HClpH 6.8
Resolving Gel 1.5M Tris-HClpH 8.8
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Sample Preparation
Ensure that sample has fully dentured!
In the Gel
In the Sample
Stacking Gel 0.5M Tris-HClpH 6.8
Laemmli Sample Buffer
0.5M Tris-HCl,pH 6.8 SDS Glycerol
Bromophenol Blue
Resolving Gel 1.5M Tris-HClpH 8.8
Boil Sample for 1-5 min
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Electrophoresis Buffer
Electrophoresis Buffer
Tris Base, Glycine, SDS
FullyProtonated
Loss of 1Proton
Loss of 2Protons
-
H2N-CH2-COO
AcidicForm
ZwitterionicForm
BasicForm
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SDS-PAGE
In the Gel
Gly lags
Stacking Gel 0.5M Tris-HClpH 6.8
-

H3N-CH2-COO
Zwitterion Form
Gly leads
-
Resolving Gel 1.5M Tris-HClpH 8.8
H2N-CH2-COO
Basic Form
Note Discontinuous SDS-PAGE is depicted here!
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Migration in an SDS-PAGE Gel
-

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Migration in an SDS-PAGE Gel
Stop electrophoresis when dye front
reaches bottom of gel
Stain with Coomassie
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Purity

• Purity is a measure of how undefiled a protein
  sample is.

Pure protein
Lots of impurities
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Yield
Example
1 2 3
After 2 steps of purification
Yield (208 / 358.2) x 100 58.1
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Protein Sequencing

• Separate subunits
• Dansyl Chloride Reaction
• Proteolytic Digestion
• Cyanogen Bromide Cleavage
• Edman Degradation

Study how each works!
Read on yourown!
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How to separate subunits?
Dithiothreitol, DTT
2-Mercaptoethanol
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Proteolytic Digestion
Know this!
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Proteolytic Digestion
How many fragments would result from digestion
with trypsin?
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Proteolytic Digestion
Trypsin cleaves after Lys (K) and Arg (R)
16 fragments!
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Protein Structure Classifications

• 1 amino acid sequence
• 2 local spatial arrangement of a polypeptide
  backbone without regard for side chains
• 3 3D structure of a protein including its side
  chains
• 4 spatial arrangements of subunits

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Tertiary Folds
Some proteins only have alpha helices (plus
turns and random coils). Others only have beta
sheets (plus turns and random coils).
Alpha
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Alpha/Beta Tertiary Folds
Some proteins have a combination of alpha helices
and beta sheets(plus turns and random coils).
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Biomolecular Stability
Nucleic acids and proteins are stabilized by the
same types of intermolecular forces.
Hydrophobic Effect the tendency of water to
minimize its contact with hydrophobic groups in
molecules. How does the hydrophobic effect
impact proteins and nucleic acids?
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Entropy
Entropy measures the spontaneous dispersal of
energy how much energy is spread out in a
process -or- how widely spread out it becomes
at a specific temperature
http//www.entropysite.com
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Entropy the Hydrophobic Effect
How is entropy increased by the hydrophobic
effect?
http//www.cryst.bbk.ac.uk/PPS2/projects/day/TDayD
iss/Major.html
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How do bond energies compare?
Type of Bond Bond Strength (kJ/mol) Covalent
348 - 460 Ionic Interaction 86 Hydrogen
Bond 20 Dipole-dipole 9.3 London
Dispersion 0.3
Table 2-1
Relatively speaking, H-bonds are weak,but they
are not nearly as weak as one might expect!
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Why Do Base Pairs Stack?
ENTROPY Hydrophobic effect induces release of
water binding to DNA bps such that the
hydrophobic ring systems can stack on top of each
other to minimize contact with water. Consider
the magnitude of stacking energy
Etc.
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Forces Stabilizing Biomolecule Structure
Proteins Nucleic Acids Hydrophobic
Effect Hydrophobic Effect Globular shape
Base Stacking Disulfide Bonds H-Bonds H-bonds
Alpha Helices Base Pairing Beta
Sheets Ionic Interactions Ionic Interactions
Salt Bridges Metal Ions