SURVEY OF
BIOCHEMISTRY
Proteins and
Biomolecular Stability
1
Protein Structure
• Primary (1°): amino acid sequence
• Secondary (2°)
– Alpha Helix
– Beta Sheet
• Tertiary (3°)
• Quaternary (4°)
2
Primary and Secondary Structure
Myoglobin - 2V1K
Superoxide Dismutase - 1XSO
3
Recap
• Structures of 20 amino acids
• pKa and pI
• 1°: Polypeptide Sequence
• 2°: Secondary Structures
– Alpha Helices
– Beta Sheets
4
Recap continued
• Protein Purification Methods
– Gel Filtration
– Ion Exchange
– Affinity
• How to assess purification?
– Purity
– Yield
5
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
6
Sodium Dodecyl Sulfate
SDS confers negative charge on proteins and denatures proteins
Amphiphilic
Hydrophilic
Hydrophobic
Anionic Detergent
in a wide variety of products
Sodium Dodecyl Sulfate
(Lauryl Sulfate)
CH3(CH2)11OSO3
Proteins are primarily
denatured by boiling them
prior to electrophoresis!
7
SDS-PAGE Buffers
Buffers maintain pH control
Allow gel to fully polymerize
Stacking Gel
0.5M Tris-HCl
pH 6.8
Resolving Gel
1.5M Tris-HCl
pH 8.8
8
Sample Preparation
Ensure that sample has fully dentured!
In the Gel
Stacking Gel
0.5M Tris-HCl
pH 6.8
Resolving Gel
1.5M Tris-HCl
pH 8.8
In the Sample
Laemmli Sample
Buffer
0.5M Tris-HCl,pH 6.8
SDS
Glycerol
Bromophenol Blue
Boil Sample for 1-5 min
9
Electrophoresis Buffer
Electrophoresis
Buffer
Tris Base, Glycine, SDS
Fully
Protonated
+ H N-CH -COOH
3
2
Acidic
Form
Loss of 1
Proton
+ H N-CH -COO 3
2
Zwitterionic
Form
Loss of 2
Protons
H2N-CH2-COO Basic
Form
10
SDS-PAGE
In the Gel
Stacking Gel
0.5M Tris-HCl
pH 6.8
Gly lags
+ H3N-CH2-COO Zwitterion Form
Gly leads
Resolving Gel
1.5M Tris-HCl
pH 8.8
H2N-CH2-COO Basic Form
Note: Discontinuous SDS-PAGE is depicted here!
11
Migration in an SDS-PAGE Gel
-
+
12
Migration in an SDS-PAGE Gel
Stop electrophoresis
when dye front reaches
bottom of gel
Stain with Coomassie
13
Purity
• Purity is a measure of how undefiled a
protein sample is.
Pure protein
Lots of impurities
14
Yield
% Yield =
Amount of protein recovered
Amount of protein initially
Example:
1
2
3
% Yield = (208 / 358.2) x 100 = 58.1%
After 2 steps
of purification
15
Protein Sequencing
• Separate subunits
Study how
each works!
• Dansyl Chloride Reaction
• Proteolytic Digestion
Read on your
own!
• Cyanogen Bromide Cleavage
• Edman Degradation
16
How to separate subunits?
Dithiothreitol, DTT
2-Mercaptoethanol
17
Proteolytic Digestion
Know this!
18
Proteolytic Digestion
How many fragments would result from digestion with
trypsin?
19
Proteolytic Digestion
Trypsin cleaves after Lys (K) and Arg (R):
16
fragments!
20
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
21
Tertiary Folds
Some proteins
only have alpha
helices
(plus turns and
random coils).
Others only have
beta sheets
(plus turns and
random coils).
Alpha
Beta
22
Alpha/Beta Tertiary Folds
Some proteins have a
combination of alpha
helices and beta sheets
(plus turns and random
coils).
23
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?
24
Entropy
Entropy measures the
spontaneous dispersal of energy:
how much energy is spread out
in a process
-orhow widely spread out it
becomes — at a specific
temperature
http://www.entropysite.com
25
Entropy & the Hydrophobic Effect
How is entropy increased by the hydrophobic effect?
http://www.cryst.bbk.ac.uk/PPS2/projects/day/TDayDiss/Major.html
26
How do bond energies compare?
Type of Bond
Covalent
Ionic Interaction
Hydrogen Bond
Dipole-dipole
London Dispersion
Bond Strength (kJ/mol)
348 - 460
86
20
Table 2-1
9.3
0.3
Relatively speaking, H-bonds are weak,
but they are not nearly as weak as one might expect!
27
Why Do Base Pairs Stack?
ENTROPY
Hydrophobic effect
induces release of
water “binding” to DNA
bp’s 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.
28
Forces Stabilizing Biomolecule
Structure
Proteins
Nucleic Acids
Hydrophobic Effect
Globular shape
Hydrophobic Effect
Base Stacking
Disulfide Bonds
H-Bonds
Alpha Helices
Beta Sheets
H-bonds
Base Pairing
Ionic Interactions
Salt Bridges
Metal Ions
Ionic Interactions
29