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1/18/2017
Amino Acids
Pratt & Cornely
Chapter 4
Amino Acid Structure
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Alpha carbon
Sidechain
Proteins
peptides
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Stereochemisty
• L‐amino acids
– Glycine
– R/S vs D/L
– L‐isoleucine
• racemization
Common Amino Acids
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Which amino acid(s)...
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•
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Is achiral?
Has a secondary amino group?
Has a chiral sidechain?
Form these derivatives:
Acid/base chemistry
• Charged amino acids
• Other amino acids are ionizable
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Ionization of Amino Acids
• Polyprotic acids
• Alanine
– pKa1 = 2.4
– pKa2 = 9.9
• Zwitterion
• Isoelectric point
• More acidic
than typical
carboxylic acid
Titration Curve
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Titration Curve
Which amino acid?
Ionization States Of Amino Acids
G
F
E
D
B
C
A
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Disulfide bond formation
Peptide bonds
• Amide bond
• Primary structure
• N‐ and C‐
terminus
• Condensation and hydrolysis
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Polypeptides
Drawing Peptides
• Sidechains
• Stereochemistry
• Ionization states
• Example: Draw the peptide AHSCVE at pH 8.
• Steps
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–
–
–
Backbone
Stereochemistry
Sidechains
Check ionization
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Example: Draw the peptide AHSCVE at pH 8.
Step 1
O
O
H
N
O
H
N
H3N
H
N
N
H
N
H
O
O
O
Step 2
O
O
O
H
N
O
H
N
H3N
H
N
N
H
N
H
O
O
O
O
Step 3
OH
CH3
O
O
H
N
H3N
N
H
H
N
O
O
SH
N
H
OH
O
pKa 4.1
pKa 8.4
Step 4
pKa 2
H
N
N
H
pKa 9
O
H
N
HO
pKa 6
O
OH
CH3
O
O
H
N
O
H
N
H3N
H
N
N
H
H
N
O
N
H
O
O
O
SH
pKa 6
N
but significantly
deprotonated
pKa 4
O
O
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Basis of secondary Structure
• Polarity
• Rigidity
• Cis/trans
Conformational Constraint
NOT cis/trans
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Dihedral Angles
Ramachandran Plots
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Ramachandran Plots
Alpha Helix
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Right handed
Polarity
n and n + 4
Gly and Pro
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Helical Wheel
• Problem 31. The sequence of a domain of the gp160 protein(HIV) is shown below using one‐letter codes for the amino acids. Plot this sequence on the helical wheel. What do you notice about the amino acid residues on either side of the wheel? MRVKEKYQHLWRWGWRWG
Helical Wheel
• The sequence of a domain of the gp160 protein(HIV) is shown below using one‐letter codes for the amino acids. Plot this sequence on the helical wheel. What do you notice about the amino acid residues on either side of the wheel? MRVKEKYQHLWRWGWRWG
W
Q
M
R
E
K
R
W
H
G
R
W
Y
G
K
V
L
W
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Beta Sheets
• Alternating sidechains can lead to amphipathic sheets
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Irregular Secondary Structure
• Nonrepeating loops and turns
• Change of direction
• Turns have about 4 residues
• Internal H‐bonds
• Gly, Pro Tertiary Structure
• Too many shapes to memorize
• But not an infinite number of possibilities
• Take away the ability to read a paper
– Discussions of motifs and why important
– Discussion of domains and why important
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Motifs (Super Secondary Structure)
• Recognizable combinations of helices, loops, and sheets
• Match
– Helix‐loop‐
helix
– Helix bundle
– Hairpin
– ‐sandwich
Studying Motifs
• Some Motifs are highly studied
• Know the lingo
– Leucine zipper
– Zinc finger
• Often have recurring applications
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Domains
• Discrete, independently folded unit (may maintain shape when cleaved on loop)
• May have separate activities: “ATP binding domain” or “catalytic domain”
• Similar activity = similar structure across many proteins
• Binding pockets at interfaces
How many domains?
Common Domains
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Major classes of globular proteins
• Mostly 
• Mostly 
• /
combination
• Little secondary
Thermodynamics of protein folding
• G might be 40 kJ/mol
for small protein (about 2 H‐bonds)
• Hydrophobic effect is important...but the most important?
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Thermodynamic Contributions to RNase
Pace, C.N. Meth. Enz. 1995, 259, 538‐554.
Other Stabilization
• Ion pair (salt bridge)
• Zinc finger
• Disulfide bond
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Protein Folding
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Denature/renature
Native structure
Domains fold separately
Global vs. local minima
Molecular chaperones
Problem 40. Proteins can be denatured by agents that alter the balance of weak noncovalent forces that maintain the native conformation. How would these agents cause a protein to denature? Heat, pH, detergent, 2‐
mercaptoethanol
Protein processing
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Thermodynamics and Kinetics
• Levinthal’s paradox: not random sampling of all possible conformations
• Energy funnel
• Series of irreversible steps
• Entropy traps
Quaternary Structure
• Multiple subunits: Oligomers
• Homodimer, heterotrimer
• Advantages
– Economy
– Stability
– regulation
2
3
22
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Protein Purification
• Chromatography
– Affinity
– HPLC
– Size Exclusion (gel filtration)
– Ion exchange
• Electrophoresis
– Isoelectric focusing
– SDS‐PAGE
Size exclusion Chromatography
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Ion Exchange Chromatography
Isoelectric Focusing
From wikipedia
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SDS PAGE
• Sodium dodecyl sulfate
• Polyacrylamide gel electrophoresis
• Analaysis
technique
• Small proteins move faster
Amino Acid Sequencing
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Purify protein
Break disulfide bonds
Enzymatic digest (protease)
Edman degradation
Overlap fragments
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Partial Digestion
Partial Digestion
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Edman
Degradation
Sequencing Oligopeptides
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X‐Ray crystallography
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