Biochemistry I
CHEM-UA 881
Fall 2023
September 14, 2023
Lecture 4:
Chapters 4.1/4.2: Peptides and Secondary Structure of
Proteins
(Introduction to Protein Folding, Chapter 4.3)
1
Solve this one on your own….
Determine the pI of the following peptide:
1. Determine the titratable functional groups.
NH2-Ala-Glu-Arg-Phe-Asp-His-COOH
2. Assign pKa values to these groups.
NH2-Ala-Glu-Arg-Phe-Asp-His-COOH
pKa: 8.0
4.1 12.5
4.1 6.0 3.1
3. Write down several hypothetical pH values and
assess the corresponding charge.
2
Textbook readings and problems for Lecture 3
Miesfeld & McEvoy (Biochemistry)
Chapter 2.2 (ionization of water; acids, bases, pKa)
Chapter 4.1 (amino acids and peptides)
Textbook Practice Problems:
Chapter 2
Challenge problems: 28-31, 34
Chapter 4
Review questions: 2, 3
Challenge problems: 15, 16, 17, 18, 19, 21, 24
3
• Alcohol
• Indole
• Phenol
amino acids,
do you need to
• Guanidinium
•For
Carboxylic
acid what
know for the quiz• next
Phenylweek and
• Amine
exams?
• Thiol
• Amide
Know these, 1 letter code, three letter code, proper spelling
of full amino acid, pKa, and charge at neutral pH.
Be able to draw most amino acids, including stereochemistry
Lecture 4 Topics
• How does primary sequence impact overall
structure?
• Flexibility of peptides (dihedral angles) and impact on
structure
• Common secondary structures in proteins (a-helices,
b-sheets and b-turns)
How do non-covalent interactions play a role
in the formation of protein structure?
5
Four “levels” of protein structure
6
Does the primary sequence of proteins
dictate their tertiary structure?
Levinthal's paradox: Do proteins fold by sampling
all possible conformations?
7
Butane can be found in its more stable conformation
100-1000X more often than in the less stable
conformations. Do proteins do the same calculations?
8
One view of the protein folding landscape
If you don’t know where you are going, you
might wind up someplace else.
-Yogi Berra
9
Levinthal's paradox: Do proteins fold by sampling all
possible conformations?
• Imagine that each amino acid consisted of only a single rotatable bond
(instead of 2*) in a protein of 101 amino acid residues.
• Imagine that there were only three possible configurations around each
of those bonds.
• This means that the protein could adopt 3100, or 5 × 1047 different
conformations.
• If the protein is able to sample 1013 different bond configurations per
second then it would take 1027 years to sample all possible
conformations of the protein. (Longer than the age of the universe!)
• Small proteins usually fold spontaneously within seconds and even the
largest proteins fold within minutes.
Adapted from: http://sandwalk.blogspot.com/2008/04/levinthals-paradox.html
10
1972 Nobel Prize in Chemistry: connection between
chemical structure and catalytic activity
Christian B. Anfinsen was awarded "for his work on ribonuclease, especially
concerning the connection between the amino acid sequence and the
biologically active conformation”; Stanford Moore and William H. Stein were
awarded "for their contribution to the understanding of the connection
between chemical structure and catalytic activity of the active centre of the
ribonuclease molecule."
11
https://www.nobelprize.org/prizes/chemistry/1972/summary/
Anfinsen’s Experiment: RNase A Folding
Anfinsen postulated that
the native structure of a
protein is the
thermodynamically
stable structure; it
depends only on the
amino acid sequence and
on the conditions of
solution, and not on the
kinetic folding route.
One of the key factors in choosing the protein to study:
RNase A contains 4 disulfide bonds
12
RNase is an enzyme
that cleaves RNA
13
Anfinsen’s Refolding Experiment I
O
What are the added reagents doing in this experiment? H N
2
Haber, Anfinsen (1961) J. Biol. Chem., 236(2): 422-4.
NH 2
urea
14
b-mercaptoethanol (BME) reduces
disulfide bonds
b-mercaptoethanol
This reaction is a disulfide exchange.
Next Question: What is the role of urea?
15
Hydrogenbonding groups
in proteins are
disrupted by
urea
O
H 2N
NH 2
urea
(this simple reasoning is likely
not the whole story:
interactions of urea with
water solvent likely have an
important impact on the
hydrophobic effect…we will
come back to this)
Urea interacts directly with
polar residues in the protein.
16
H-bonds between residues in proteins help
stabilize 3D structure
17
RNAse refolds after removal of denaturing agent
18
Except….RNAse didn’t refold into its native
state after removal of denaturing agent
until….
Trace amounts of
b-mercaptoethanol
19
Q. Why is trace mercaptoethanol
needed to refold?
• RNAse refolds after removal
of denaturing agent.
• If it is oxidized in the
presence of urea the protein
forms incorrect R-S-S-R
bonds, but they find the
native structure in the
presence of a trace of thiol
reagent.
20
Overall: Anfinsen’s Refolding Experiment II
21
Anfinsen's work showed convincingly that proteins
can indeed adopt their native conformation
spontaneously, i.e. sequence determines structure.
This idea informs our understanding of protein folding
(and therefore protein structure):
22
Anfinsen's work showed convincingly that proteins
can indeed adopt their native conformation
spontaneously, i.e. sequence determines structure.
This idea informs our understanding of protein folding
(and therefore protein structure):
23
?
24
Simulation of millisecond protein folding: NTL9
https://www.youtube.com/watch?v=gFcp2Xpd29I
25
Proteins are composed of polypeptide chains
26
Peptide conformation is defined by 3 dihedral angles
R
N
H
O
H
N
O
R
R
N
H
H
N
O
omega (w)
R
O
H
N
φ
O ω
N
H
ψ
R
R
27
A dihedral angle is the angle between
two planes
Also called a torsional angle
28
The amide (peptide) bond is rigid
resonance!
O
R
DG#= 23 kcal/mol
O
N
H
R
R
N
H
R
Peptide bond: 1.32 Å
C-N: 1.45 Å
C=N: 1.25 Å
O
R
+2.3 kcal/mol
O
R
N
H
trans-amide
R
H
N
R
cis-amide
29
The peptide bond is constrained in either the trans or
cis configuration (isomers)
Cis
Trans
Trans is more stable by ~8 kJ/mol (~2 kcal/mol)
Why? (is it just steric interference?)
30
Proline residues in proteins are found ~10%
Cis conformation
Trans Pro
Cis Pro
Ca-N bond
Difference is ~ 1.0 kcal/mol
Many occur in b-turn structures
31
What about the f and y bond rotations?
for f
O ω
O
N
H
ψ
R
H
N
φ
>(
>(
R
for y
R
You can think of f and y dihedrals using the same
principles as for substituted ethane or butane
You will be given a detailed dihedral angle problem in recitation. 33
Phi (Φ) angle
2
1
1
3
2
3
4
C'—N—Cα—C'
4
The phi angle is between the
plane defined by atoms 1-3,
and the plane from atoms 2-4.
Clockwise rotation is positive.
-90°
ϕ≈ -70°
34
Psi (ψ) angles
1
1
2
2
3
4
N—Cα—C'—N
3
4
The psi angle is between the
plane defined by atoms 1-3,
and the plane from atoms 2-4.
Clockwise rotation is positive.
ψ ≈ +40o
90°
35
Rotation about dihedral angles create
different structures in proteins
• In principle Φ and ψ could have any value between -180° and
180°
• However, many values are prohibited by steric interference
between atoms in the polypeptide backbone and amino acid
side chains
36
A Ramachandaran Plot indicates
“allowed” dihedral angles (1963)
G N Ramachandran
“steric exclusion”
The plot shows accessible (low energy) f and y conformations. Dark areas
are “allowed”, white areas denote high energy conformations.
Possible conformations determined based on VDW radii and dihedral angles
37
Real Data from 207 Protein Structures:
Calculate using PDB structures and a calculator (code) or from one of several
38
programs (e.g., Pymol)
Q. One of these plots is for glycine and
the other for alanine.
a)
b)
39
40
Certain dihedral angles represent
specific protein secondary structures
41
Average dihedral angles for various 2° structures
42
Main types of secondary structure are found
in proteins
1. Helix
2. Sheet
3. Loops/Random coil
43
Secondary structure refers to regular folds
of the backbone
• More than 30% of the residues in proteins have
a-helical values of f and y
• Next most common are b strands
Estrogen
Receptor
(ligand
binding
domain)
Green
Fluorescent
Protein
(GFP)
44
The a-helix is a coiled structure stabilized by H-bonds
Residue “i” C=O makes Hbond with residue “i+4" N-H
Pitch is the
vertical
distance
between
one
consecutive
turn
All N-H and C=O pairs make
H-bonds except the first 3
N-H (at N-terminus) and last
three C=O (at C-terminus)
Distance
between
adjacent
residues
in the helix
45
Almost all a-helices in proteins are right-handed
• Right-handed helices are energetically more favorable (less steric clash
between side chains and the backbone).
• Almost all a-helices found in proteins are right-handed
46
Like amino acids, a-helices have dipole moments
Another view of this…
N-term
C-term
47
The structure of the a-helix* was
predicted before it was confirmed
Linus Pauling (Chemist)
Robert Corey (X-ray crystallographer)
Herman Branson (Physicist)
*“Biochemists will note that the C=O groups of the a-helix
point in the direction of its C terminus”..but...“the a-helix
shown is left-handed and made up of D-amino acids.”
Pauling, L., Corey, R. B. & Branson, H. R. Proc. Natl. Acad. Sci. USA 1951,
37, 205; Eisenberg, D. Proc. Natl. Acad. Sci. USA 2003, 100, 11207.
a-helix model
depicted
in their
1951 paper
48
The i, i+3 and i+4 residues reside on the same
face of the a-helix
1. Side chains at i position can interact
with side chains at i+3/i+4 positions using
salt-bridges, hydrogen bonding, pistacking or van der Waals interactions
i+7
i+4
i+3
i+2
i
i+3
i+1
i+6
i
Recall: 3.6 residues per helical
turn
Phosphofructokinase-1 (145-157) i + 10
2. Many a-helices are amphipathic;
one side is hydrophobic, the other is
hydrophilic.
49
In a helical wheel diagram, we look down the
central axis of the helix
Phosphofructokinase-1 (145-157)
• Start with amino acid 1 at the top of the
wheel (T)
• Each residue is placed sequentially around
the wheel (100° from the previous residue)
• Decreasing diameters indicates amino acids
that are further away from the initial
residues of the helix
Schiffer, M.; Edmundson, A. B. Biophys. J. 1967, 7, 121.
50
Sequence is determined by where the helix is located in a protein
BURIED
PARTIALLY EXPOSED
Hydrophobic in yellow; red: (-) charge ; blue: (+) charge
FULLY EXPOSED
51
Science 1990, 250, 669.
52
Some residues prefer to be in the helical
conformation more than others
1. Helix propensity is favored by Ala, destabilized by
Gly or Pro.
2. Leu stabilizes helix more than I or V. Crowding Cb is
less favorable.
O
H 2N α
β
Leu
O
O
OH
H 2N α
β
γ
Ile
OH
H 2N α
O
OH
H 2N α
OH
β
β
γ
S
Met
Val
3. Side chains interact at spacing of i,i+3 and i,i+4: acid
base bridges (E,D to K,R) stabilize helix, hydrophobic
groups too.
53
Pro and Gly disfavor alpha-helix formation
Why?
Higher propensity
Helix breakers
54
Stabilized helices are used as therapeutics
“Ozempic® (semaglutide) injection 0.5 mg, 1 mg,
or 2 mg is an injectable prescription medicine
used:
• along with diet and exercise to improve blood
sugar (glucose) in adults with type 2 diabetes.
• to reduce the risk of major cardiovascular
events such as heart attack, stroke, or death in
adults with type 2 diabetes with known heart
disease.”
semaglutide
J. Med. Chem. 2015, 58, 18, 7370.
https://www.ozempic.com/
• Unnatural peptide
(peptidomimetic)
• Glucagon-like peptide-1 (GLP-1)
analog
• Lowers blood glucose
GLP-1 receptor extracellular domain
55
b-Strand/b-Sheet Secondary Structure
Consecutive dihedral angles of
(Φ, ψ) of -120° and 120°
• Often twisted rather than
completely planar
• Distance between adjacent
amino acids ~ 3.4 Å
• Enriched in Val, Ile, Thr
(branched amino acids);
Tyr, Trp, Phe (aromatic); Cys
•
Ribbon style: b-strand has arrowhead
on the C-terminus of the helix.
56
b-Strands come together in b-Sheets:
Parallel Sheet Arrangement
N!C
N!C
What do you notice about the H-bonds between the strands?
57
b-Strands come together in b-Sheets:
Antiparallel Sheet Arrangement
C"N
N!C
• Hydrogen bonds line up between the two strands
• Antiparallel b-sheets are more common in proteins
due to increased stability
58
b-Sheets are pleated
59
Like helices, b-strands can be amphipathic
POLAR FACE
HYDROPHOBIC
FACE
Often see HPHP (H-hydrophobic, P-polar) pattern in strands
(different from the spacing of this pattern in helices)
60
Short Loops (b turns) Can Connect Adjacent b-Strands
turn: reverse the direction
Connect 2 b-strands
in antiparallel b-sheets
Q: Gly and Pro
are common
in turns..why?
Type I
Type II
Type I and type II b turns differ in the orientation of the peptide plane
between the second and third amino acid residues in the turn.
61
Silk: A Protein-Based Material
Silk is a fibrous protein consisting of three subunits, one of which
consists almost entirely of b-sheets, which collectively give silk its
strength.
The silk used in fabric comes from
the cocoon of the Japanese
silkworm Bombyx mori.
Textbook readings and problems for Lecture 4
Miesfeld & McEvoy (Biochemistry)
Chapter 4.1: peptides (torsional angles)
Chapter 4.2: secondary structures
Chapter 4.3: protein folding
Textbook Practice Problems:
Chapter 4
Review Problems: 7, 9
Challenge Problems: 23, 26-28
The structures, names and/or abbreviations of amino acids
will be on the quiz next week.
63