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SURVEY OF
BIOCHEMISTRY

Amino Acids and Proteins

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Amino Acids
Can you give the 1-letter and 3-letter names
for all 20 amino acids within 5 minutes?

Can you draw a oligopeptide of any given
sequence?
With correct stereochemistry?
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Amino Acids:
Nonpolar Side Chains
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Amino Acids:
Polar, Uncharged Side Chains
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Cysteine & Disulfide Bonds
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Cysteine & Disulfide Bonds
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Amino Acids:
Polar, Uncharged Side Chains
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Amino Acids:
Polar, Uncharged Side Chains
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Amino Acids:
Polar, Charged
Be able to draw,
name and give
1-letter/3-letter
codes for
all 20
amino acids!
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pK
a
and pI
pI = Isoelectric point
the pH at which a molecule carries no
net electric charge
pK
a1
pK
a2

pI = [pK
a1
+ pK
a2
]

1
2
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Polypeptide Sequence
Nomenclature
Tetrapeptide: AYDG
Be able to draw
a polypeptide!
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Peptide Bond
Peptide bond links one amino acid to another.
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Torsion Angles in Proteins

Psi()
Phi()
Peptides
typically
assume
the trans
conformation
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Torsion Angles Defined
Psi: angle made from atoms
N-C
alpha
-C
carbonyl
-N
n+1

N
H
N
N
O O
O H
H
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Torsion Angles Defined
Phi: angle made from atoms
C
carbonyl,n-1
-N
n
-C
alpha
-C
carbonyl,n
N
H
N
N
O O
O H
H
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Alpha Helix
Sequence coils in a right-handed
manner.

Notice hydrogen bonding along the
helical axis from carbonyl oxygen
(of residue n) to the amino hydrogen
of residue (n+4).

Hydrogen bonding stabilizes the
alpha helical structure!
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Beta Sheets
Notice the
hydrogen
bonding
from the
carbonyl
oxygen to the
amino
hydrogen
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Protein Purification Overview
Courtesy of
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Salting Out: (NH
4
)
2
SO
4

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Gel Filtration Chromatography
Separation based on Size
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Ion Exchange Chromatography
Separation based on Charge
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Affinity Chromatography
Affinity
What could be
to cause proteins
to elute off of an
affinity column?
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Ways to Assess Protein
Purification
Total Protein Concentration Assays
Beers Law
Colorimetric Assays

Specific Protein Assays
Activity Assays
Immunoassays
ELISA
RIA
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Beers Law

Abc
What is the molar concentration of a solution of
Bovine Serum Albumin (BSA) that exhibits an
A
280
of 0.75 with a path length of 1 cm?
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Beers Law

Abc
What is the molar concentration of a solution of
Bovine Serum Albumin (BSA) that exhibits an
A
280
of 0.75 with a path length of 1 cm?
Conc. =
0.75
(43,824 M
-1
cm
-1
) x (1 cm)
Conc. = 0.00001711 M = 17 M
Must know
the value of

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Bradford Protein Assay
Suppose you have a protein mixture and you need to
determine the protein concentration.

You cannot use Beers Law.
Because you would not know the extinction coefficient
for the protein mixture at 280 nm
Alternative approach:
Bradford Protein Assay
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Bradford Protein Assays
How is data generated?
How is data analyzed?
Bradford Protein Assay reagent
contains Coomassie brilliant blue
which reacts with basic (esp.
Arg) and aromatic amino acids
to yield a blue color with intensity
proportional
to the protein concentration.
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Data Generation: Bradford Assay
Reference contains only buffer
Create a series of protein
standards at known increasing
concentrations and mix with
the
Bradford reagent
2.5 5 10 15 20 25 g/mL
Treat the protein mixture of unknown concentration
in the same manner as the standards and compare -
visually and spectrophotometrically!
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Bradford Assay Data Analysis
Graph the absorbance
at 595 nm as a function
of [standard] in g/mL.

Fit the line and use it
to deduce the [protein]
in the mixture based on
its absorbance.