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8/12/2019 Cellular peptides
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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 give1-letter/3-letter
codes for
all 20
amino acids!
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pKaand pI
pI = Isoelectric pointthe pH at which a molecule carries nonet electric charge
pKa1pKa2
pI = [pKa1+ pKa2]1
2
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Polypeptide SequenceNomenclature
Tetrapeptide: AYDGBe able to drawa 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 transconformation
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Torsion Angles Defined
Psi: angle made from atomsN-Calpha-Ccarbonyl-Nn+1
NH
NN
O O
OH
H
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Torsion Angles Defined
Phi: angle made from atomsCcarbonyl,n-1-Nn-Calpha-Ccarbonyl,n
NH
N
NO O
OH
H
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Alpha Helix
Sequence coils in a right-handed
manner.
Notice hydrogen bonding along thehelical axisfrom 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 thecarbonyl
oxygen to the
amino
hydrogen
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Protein Purification Overview
Courtesy of
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Salting Out: (NH4)2SO4
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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
AffinityWhat could beto cause proteins
to elute off of an
affinity column?
W t A P t i
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Ways to Assess ProteinPurification
Total Protein Concentration AssaysBeers Law
Colorimetric Assays
Specific Protein Assays
Activity Assays
Immunoassays ELISA
RIA
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Beers Law
A bcWhat is the molar concentrat ion o f a solut ion of
Bovine Serum Album in (BSA) that exhibi ts anA 280of 0.75 w ith a path leng th o f 1 cm?
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Beers Law
A bcWhat is the molar concentrat ion o f a solut ion of
Bovine Serum Album in (BSA) that exhibi ts anA 280of 0.75 w ith a path leng th o f 1 cm?
Conc. =0.75
(43,824 M-1cm-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 increasingconcentrations and mix with
the
Bradford reagent2.5 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.