Download survey of biochemistry - School of Chemistry and Biochemistry

SURVEY OF
BIOCHEMISTRY
Amino Acids and Proteins
1
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?
2
Amino Acids:
Nonpolar Side Chains
3
Amino Acids:
Polar, Uncharged Side Chains
4
Cysteine & Disulfide Bonds
5
Cysteine & Disulfide Bonds
6
Amino Acids:
Polar, Uncharged Side Chains
7
Amino Acids:
Polar, Uncharged Side Chains
8
Amino Acids:
Polar, Charged
Be able to draw,
name and give
1-letter/3-letter
codes for
all 20
amino acids!
9
pKa and pI
• pI = Isoelectric point
the pH at which a molecule carries no
net electric charge
pKa2
pKa1
1
pI =
[pKa1 + pKa2]
2
10
Polypeptide Sequence
Nomenclature
Be able to draw
a polypeptide!
Tetrapeptide: AYDG
11
Peptide Bond
Peptide bond links one amino acid to another.
12
Torsion Angles in Proteins
Psi()
Phi()
Peptides
typically
assume
the trans
conformation
13
Torsion Angles Defined…
• Psi: angle made from atoms
N-Calpha-Ccarbonyl-Nn+1
N
H
H
N
O
O
N
H
O
14
Torsion Angles Defined…
• Phi: angle made from atoms
Ccarbonyl,n-1-Nn-Calpha-Ccarbonyl,n
N
H
H
N
O
O
N
H
O
15
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!
16
Beta Sheets
Notice the
hydrogen
bonding
from the
carbonyl
oxygen to the
amino
hydrogen
17
Protein Purification Overview
Courtesy of
18
Salting Out: (NH4)2SO4
19
Gel Filtration Chromatography
Separation based on Size
20
Ion Exchange Chromatography
Separation based on Charge
21
Affinity Chromatography
Affinity
What could be
to cause proteins
to elute off of an
affinity column?
22
Ways to Assess Protein
Purification
• Total Protein Concentration Assays
– Beer’s Law
– Colorimetric Assays
• Specific Protein Assays
– Activity Assays
– Immunoassays
• ELISA
• RIA
23
Beer’s Law
A  bc
What is the molar concentration of a solution of
Bovine Serum Albumin (BSA) that exhibits an
A280 of 0.75 with a path length of 1 cm?
24
Beer’s Law
A  bc
What is the molar concentration of a solution of
Bovine Serum Albumin (BSA) that exhibits an
A280 of 0.75 with a path length of 1 cm?
Conc. =
0.75
(43,824 M-1cm-1) x (1 cm)
Conc. =
0.00001711 M = 17 µM
Must know
the value of

25
Bradford Protein Assay
Suppose you have a protein mixture and you need to
determine the protein concentration.
You cannot use Beer’s Law.
Because you would not know the extinction coefficient
for the protein mixture at 280 nm
Alternative approach:
Bradford Protein Assay
26
Bradford Protein Assays
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.
How is data generated?
How is data analyzed?
27
Data Generation: Bradford Assay
Reference contains only buffer
2.5
5
10
15
20
25 µg/mL
Create a series of protein
standards at known
increasing concentrations
and mix with the
Bradford reagent
Treat the protein mixture of unknown concentration
in the same manner as the standards and compare visually and spectrophotometrically!
28
Bradford Assay Data Analysis
Graph the absorbance
at 595 nm as a function
of [standard] in µg/mL.
Fit the line and use it
to deduce the [protein]
in the mixture based on
its absorbance.
29