Biochemistry 2280A/2288A – Fall 2020

Topic 4: Protein
Function

Dr. Michael Boffa

 Each topic (except Topic 1) has a quiz at the end.
Course Map Your grade will include your best 20 quiz results.

UNIT A: Proteins UNIT B: Metabolism UNIT C: Nucleic Acids

01 The Chemical Basis of Life 06 Lipids & Biological Membranes 14 Nucleic Structure

02 Amino Acids 07 Carbohydrate Structure 15 DNA Replication

08 Fundamental Concepts in
03 Protein Structure & Analysis 16 DNA Repair
Metabolism

04 Protein Function 09 Carbohydrate Metabolism 17 Molecular Basis of Cancer

05 Enzyme Catalysis 10 Citric Acid Cycle 18 Prokaryotic Transcription

▪ Mid Term 11 Oxidative Phosphorylation 19 Eukaryotic Transcription

12 Lipid Metabolism 20 RNA Processing

13 Summary of Metabolic Pathways 21 Translation

▪ Mid Term 22 Recombinant DNA Technology

23 Sequencing Genomes

▪ Final Examination
Course Map

UNIT A: Proteins

01 The Chemical Basis of Life

02 Amino Acids

03 Protein Structure & Analysis

A PTM’s and Ligand Binding
04 Protein Function
B Myoglobin
05 Enzyme Catalysis
C Hemoglobin
▪ Mid Term
D Collagen
Objectives
By the end of this topic, students will be able to:

1. Describe what ligands are and how they bind to

proteins.

2. Explain the concept of Dissociation Constant (K).

3. Define, in mathematical terms, affinity and the molecular

interactions that allow proteins to interact with ligands.

4. Describe the structure and function of myoglobin,

hemoglobin and collagen in terms of the four levels of
protein structure.
 Topic 4: Protein Function
• Underlies almost everything that happens in a cell
• Including variants and PTM’s: Several hundred
thousand unique proteins in our bodies!
Name Function Example
Enzyme catalyzes covalent bond breakage or formation protein kinase
Structural provides mechanical support to cells & tissues keratin
Transport carries small molecules or ions hemoglobin
Motor generates movement in cells and tissues myosin
Storage stores small molecules or ions ferritin
Signal carries signals from cell to cell insulin
Receptor detects signals and transmits them to the cell Trk (NGF)
Gene Regulator binds to DNA to switch genes on or off p53
Post-translational modifications
Phosphorylation, Ubiquitination, Acetylation,
Sumoylation → alters stability or signaling

Glycosylation → affects protein folding,

secretion, solubility, binding to other
biomolecules

Myristoylation, Farnesylation → alters

location

‘Lipoproteins' bind lipids

‘Metalloproteins' bind metal ions

‘Hemoproteins' have an attached heme group

Key PTM: phosphorylation
 Ligand-binding proteins
• Many proteins contain sites to which ligands specifically
bind and form a “complex” with the protein
• Ligand: molecule that can form this complex
• Binding occurs by multiple weak or a few strong forces
• leads to extreme specificity

Naproxen (Aleve ®) bound to COX-2

http://www.nature.com/nchembio/journal/v7/n11/abs/nchembio.663.html
 Ligand binding – The Dissociation
Constant
• Kd (dissociation constant) is a measure of the
strength or affinity of an interaction
• An equilibrium constant
• For a protein (P) binding ligand (L):

[P] [L]
PL P + L Kd = [PL]
Kd
PL, P, and L represent molar concentrations of the
complex, protein, and ligand, respectively.
Dissociation constants

• High affinity = tight binding = small Kd

• Units are moles/L (M)
• Biological examples:
• Calmodulin/Calcium - 0.1 μM (10-6 M)
• antibody/antigen – μM to pM (10-12 M)
• Estrogen/Estrogen receptor – μM
• biotin-streptavidin 1.0 fM (10-15 M)
Lower
Affinity

Higher
Affinity
Course Map

UNIT A: Proteins

01 The Chemical Basis of Life

02 Amino Acids

03 Protein Structure & Analysis

A PTM’s and Ligand Binding
04 Protein Function
B Myoglobin
05 Enzyme Catalysis
C Hemoglobin
▪ Mid Term
D Collagen
Objectives
By the end of this topic, students will be able to:

1. Describe what ligands are and how they bind to proteins.

2. Explain the concept of Dissociation Constant (K).

3. Define, in mathematical terms, affinity and the molecular

interactions that allow proteins to interact with ligands.

4. Describe the structure and function of myoglobin,

hemoglobin and collagen in terms of the four levels of
protein structure.
Readings

• Essential Biochemistry
• Section 5.1 (Myoglobin and Hemoglobin: Oxygen-Binding
Proteins)
 Another ligand-binding protein:
Myoglobin
• Consists of 8 a-helices plus one
heme prosthetic group
• Oxygen-binding protein
• Facilitates oxygen diffusion through
tissues

Heme
FIGURE 5.1b
 Another ligand-binding protein:
Myoglobin
• Consists of 8 a-helices plus one
heme prosthetic group
• Oxygen-binding protein
• Facilitates oxygen diffusion through
tissues
His E7

O2

His F8
FIGURE 5.1b
FIGURE 5.2
 O2 binding by myoglobin

Consider the chemical equation for O2 reversibly binding with Mb:

The dissociation constant for this reaction is:

K=
MbO2 
MbO 2 

But this relationshipis not helpful experimentally, so we need to reformulate it

in terms that we can measure.
 O2 binding by myoglobin

Begin by writing an equation that gives us a measure of the fraction of

binding sites that are occupied:
YO 2 =
MbO 2 
Mb+ MbO 2 
After some algebra, obtain:

YO =
O2 
 2
K + O2 
O2 is a gas, therefore its concentration can be expressed via its partial
pressure, pO2
 pO 2
YO 2 =
K + pO 2
 O2 binding by myoglobin

pO 2
YO 2 =
K + pO 2
When K = pO2
… half the binding sites
are occupied.

K = p50
The O2 pressure at
which Mb is 50% saturated

p50 = 2.8 torr

FIGURE 5.3
pO2 = 30 – 100 torr K = p50
Course Map

UNIT A: Proteins

01 The Chemical Basis of Life

02 Amino Acids

03 Protein Structure & Analysis

A PTM’s and Ligand Binding
04 Protein Function
B Myoglobin
05 Enzyme Catalysis
C Hemoglobin
▪ Mid Term
D Collagen
Objectives
By the end of this topic, students will be able to:

1. Describe what ligands are and how they bind to proteins.

2. Explain the concept of Dissociation Constant (K).

3. Define, in mathematical terms, affinity and the molecular

interactions that allow proteins to interact with ligands.

4. Describe the structure and function of myoglobin,

hemoglobin and collagen in terms of the four levels of
protein structure.
Readings

• Essential Biochemistry
• Section 5.1 (Myoglobin and Hemoglobin: Oxygen-Binding
Proteins)
• Section 5.2 (Hemoglobin Variants)
 Hemoglobin
a b

FIGURE 5.4

a-globin
b-globin
a myoglobin
b

identical in a- and b-globin

identical in human Mb, a- FIGURE 5.5

and b-globin

identical in all vertebrate

Mb, a- and b-globin
O2 binding by hemoglobin: cooperative!

FIGURE 5.7
 T- to R-state transition of hemoglobin
T-state
R-state
FIGURE 5.8

T-state R-state
(low affinity) (high affinity)

R-state

T-state
FIGURE 5.9
 The Bohr effect and role of BPG

stabilizes
T-state

pH 7.6 pH 7.2
FIGURE 5.10

Bohr protons
(when bound,
decrease
affinity for O2) FIGURE 5.11
O2 binding by hemoglobin

FIGURE 5.7
Hemoglobin variants causing disease (1)
 Hemoglobin variants causing disease (2)

Sickle-cell anemia:
1 of 300 known variants of
Hb.
Mutation E6V in b-chains
results in 2 fewer –ve
charges.
V creates a site for
intermolecular
hydrophobic interactions.
Objectives
By the end of this topic, students will be able to:

1. Describe what ligands are and how they bind to proteins.

2. Explain the concept of Dissociation Constant (K).

3. Define, in mathematical terms, affinity and the molecular

interactions that allow proteins to interact with ligands.

4. Describe the structure and function of myoglobin,

hemoglobin and collagen in terms of the four levels of
protein structure.
Course Map

UNIT A: Proteins

01 The Chemical Basis of Life

02 Amino Acids

03 Protein Structure & Analysis

A PTM’s and Ligand Binding
04 Protein Function
B Myoglobin
05 Enzyme Catalysis
C Hemoglobin
▪ Mid Term
D Collagen
Objectives
By the end of this topic, students will be able to:

1. Describe what ligands are and how they bind to proteins.

2. Explain the concept of Dissociation Constant (K).

3. Define, in mathematical terms, affinity and the molecular

interactions that allow proteins to interact with ligands.

4. Describe the structure and function of myoglobin,

hemoglobin and collagen in terms of the four levels of
protein structure.
Readings

• Essential Biochemistry
• Section 5.3 (Structural Proteins)
• Box 5.B
• Box 5.C
 Collagen

• Most common protein in animals

(about 25-30%)
• Extracellular, forms strong fibers or
sheets
• Connects and strengthens tissues
• Bone matrix, tendons, connective
tissue, skin
• Family of proteins, numbered I to
XIV
• Characteristic structure triple
helical
http://www.anatomybox.com/page/54/
http://wellcomeimages.org/
Collagen in the marketplace
 X3

Fig 4-29. Essential Cell Biology 4th Edition. Garland Science 2014
 Collagen helical folding

• Presence of so much proline prevents alpha helix

• Instead, forms a “poly-proline type II” helix

• More extended than a-helix
• No intra-chain H-bonds
• Amide nitrogen and oxygen atoms too far apart
• Stabilized by steric repulsion of proline side chains
• 3 residues per turn

• Collagen “triple helix” consists of 3 intertwined

polyproline type II helices
 Collagen triple helix
• Collagen has a
characteristic sequence Pro
• Every third residue is
G - ESSENTIAL Hyp
• Lots of proline and
hydroxy-proline
Gly
• Modified amino acid
with hydroxyl group
• The hydroxylation of
proline residues plays
a role in triple helix
stabilization of
collagen molecules
• Hydroxy-lysine: OH
groups serve as sites
of sugar addition.
• Lysines are also
involved in a cross-
linking reaction that Pro-collagen
creates covalent triple helix
bonds between
collagen fibrils
 Collagen triple helix
• Collagen has a
characteristic sequence
• Every third residue is
G - ESSENTIAL
• Lots of proline and
hydroxy-proline
• Modified amino acid
with hydroxyl group
• The hydroxylation of
proline residues plays
a role in triple helix
stabilization of
collagen molecules
• Hydroxy-lysine: OH
groups serve as sites
of sugar addition.
• Lysines are also
involved in a cross-
linking reaction that
creates covalent
bonds between
collagen fibrils
 Collagen triple helix
• Collagen has a
characteristic sequence Pro
• Every third residue is
G - ESSENTIAL Hyp
• Lots of proline and
hydroxy-proline
Gly
• Modified amino acid
with hydroxyl group
• The hydroxylation of
proline residues plays
a role in triple helix
stabilization of
collagen molecules
• Hydroxy-lysine: OH
groups serve as sites
of sugar addition.
• Lysines are also
involved in a cross-
linking reaction that Pro-collagen
creates covalent triple helix
bonds between
collagen fibrils
 Collagen properties

• Gives a thin (1.5 nm), long (300 nm), very strong molecule
• Quite hydrophobic, insoluble due to many exposed
hydrophobic side chains
• Must be first synthesized as precursor containing pro-peptides
which keeps it soluble→called 'pro-collagen’
• These 'pro-peptides’ increase solubility and prevent premature
fibre formation

http://jcb.rupress.org/content/144/5/1069/F1.expansion.html
 Collagen synthesis and assembly

• Pro-collagen secreted from cell

• Pro-peptides do not form triple helical structures
• Pro-peptides removed before fibril assembly
• 'pro-peptidase' enzymes present outside cells
• Collagen molecules are covalently cross-linked in
the fibril
• Lysine residues in different chains
• Generated by enzyme “lysyl oxidase”
• Gives further strength to the fibril
Collagen
synthesis
and
assembly

Glycosylation
facilitates
extracellular
solubility as well
as water
absorption.
Lysyl cross-links in collagen
 Collagen-related disease
• Scurvy
• Lack of vitamin C (ascorbic acid) in diet
• Vit C is needed for enzyme that hydroxylates Proline
• Non-hydroxylated prolines = unstable collagen
• Osteogenesis imperfecta (“bad bone”)
• Often glycine mutations
• Prevent proper assembly of triple helix
• Leads to lack of collagen
• Malformed or absent bone
 Ehlers–Danlos syndrome (EDS)
• Group of inherited connective tissue disorders
• Caused by a defect in the synthesis of collagen
• Can be caused by mutations in collagen genes themselves or processing
enzymes such as procollagen I N-proteinase or lysyl oxidase

Normal skin EDS patient skin

Niccolò Paganini (1782

46 – 1840)