Lecture 3

BME2106 - Introduction to
Cellular and Biomolecular

Engineering
Week 4
Dr. Bee Luan KHOO

blkhoo@um.cityu.edu.hk
Room no 6720
Department of Biomedical Engineering

BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong
Protein biomarkers
What is a protein?
3-Dimensional Structure of
Protein
4 levels of organization:
a. Primary structure
b. Secondary structure
c. Tertiary structure
d. Quaternary structure
Primary Structure
• The sequence of amino acid
residues which are covalently linked
by peptide bonds in a polypeptide.
• The primary structure of a protein
determines identity (fingerprint).
• Each protein species differs from
all others in its primary structure.
• The primary structure also dictates
the folding of the polypeptide
chain.
Where does the amino acid come from?
Characteristics of the Primary
Structure of a Protein
• Each protein has its unique sequences
• Not all amino acids occur in all protein; e.g. tryptophan does
not occur in RNase
• There is no rules regulating the occurrences and fix ratio to
each other, i.e. no repeating units
• Insoluble proteins have repeating unit and large amount of
hydrophobic amino acids
• Protein from homologous species has similar sequences
• Related proteins in same species have similar sequence
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BME2106 - Introduction to Cellular and Biomolecular Engineering
AA&Prtn-II BME, City University of Hong Kong
Secondary Structure
• It is the conformation of the polypeptide
which results to coiling or stacking of the
primary structure.
• It is about the micro-folding of protein.
• Major stabilizing force: H-bonds between
components of adjacent peptide bonds.
• A polypeptide tends to form secondary
structures because of the regularity of the
backbone of the chain and because the
secondary structures maximize the number
of H bonds that can be formed.
Secondary Structure
• In a typical protein, more than 60% of
the amino acid residues are involved
in 3 types of secondary structures,
namely:
-- helices,
-- pleated sheets
-- reverse turns (bends)
• These structures owe their existence
not only to the hydrogen bonds of
polypeptide chain, but also to stearic
limitations on the rotations of bonds in
the polypeptide chain.
Secondary Structure in Protein
• The three most abundant regular secondary

structures actually found in proteins are :
-- -helix,
-- parallel -pleated sheet
-- antiparallel -pleated sheet (the pleated sheets
being constructed of polypeptide chains that are
in a zig-zag conformation)
Various Forms of Secondary
Structures of Polypeptides
Model of an α-helix.
Interchain H bonds are represented by
dotted lines. The aa side chains are
represented by solid spheres.
The α-helix is a regular right-handed
helix in which the NH group of each
peptide bond is bonded to the CO group
of the residue located 4 positions later in
the chain
Termination of helical structure:
whenever proline or aa with large side
chains.
Various Forms of Secondary
Structures of Polypeptides
Model of an α-helix.
Interchain H bonds are represented by
dotted lines. The aa side chains are
represented by solid spheres.
The α-helix is a regular right-handed
helix in which the NH group of each
peptide bond is bonded to the CO group
of the residue located 4 positions later in
the chain
4 of these make 1 of this Termination of helical structure:

whenever proline or aa with large side
chains.
Various Forms of Secondary Structures
of Polypeptides
Model of β-pleated sheet:
a fully extended structure
A β-parallel pleated sheet formed by 2 adjacent polypeptide
chains. To emphasize the path taken by the polypeptide chain,
the backbone has been superimposed on a ribbon.
Note: H bonds that hold the sheet together are

perpendicular to the polypeptide backbone. Side
chains are projected above and below the surface
A β-turn allows the polypeptide

chain to bend back on itself.
Tertiary Structure
• Tertiary structure refers to the
three‐dimensional arrangement of
all the atoms that constitute a protein
molecule.
• Relates the precise spatial
coordination of secondary structure
elements and the location of all
functional groups of a single
polypeptide chain
• It is the conformation of a
polypeptide which results from
folding or supercoiling of the
secondary structure.
Tertiary Structure
• Major stabilizing
force:
Interaction
between/among
R groups of the
amino acid
residues.
• The R-groups interact
in a polypeptide chain
to create
the tertiary structure of
a protein.
Tertiary Structure
• The S-S bonds formed by the
oxidation of two sulphydryl
groups are covalent.
• The other interactions are

non-covalent. These may by
either Apolar, i.e.
hydrophobic, or Polar
i.e. hydrogen-bonding and
ionic.
Quaternary Structure
• It is the arrangement of polypeptide chains in relation to
one another in a multiple chained protein.
• Major stabilizing force: Interaction between/among R
groups of the amino acid residues.
• Dimers are associations of 2 subunits
• Tetramers are associations of 4 subunits, each subunit
being a monomer.
The quaternary structure of a globular protein composed of two

units. It becomes a functional protein only when it is a dimmer.
Structural Elements of Protein
Conformation
• Conformation - the degree of coiling or folding
Protein Conformation
• Conformation refers to the secondary and tertiary
structure jointly, that is, the folding pattern of the
polypeptide chain and all contacts between amino acid
side chains of that polypeptide chain.
• Many proteins, after being unfolded artificially

(denatured), will refold spontaneously to their original
(native) conformation when incubated at an appropriate
temperature, pH, and ionic strength.
BME2106 - Introduction
AA&Prtn-II to Cellular and Biomolecular Engineering BME, City University of Hong Kong
Ways of Maintaining Protein
Conformation
1. Noncovalent interactions are primarily responsible for
maintaining protein conformation.
2. Polypeptides in solution fold to minimize free energy.
3. Why is it important - Is the protein stable enough to function under
harsh conditions of temperature or solvent?
4. If a protein unfolds reversibly it may be fully unfolded and inactive at high
temperatures, but once it cools to room temperature, it will refold and
fully recover activity.
5. From a functional standpoint this may be all that is required for it to be
classified as thermostable. However, from a thermodynamic standpoint
(and in terms of this dissertation) it is classified as non-thermostable.
Gibbs free energy
Where T is the temperature in Kelvin.
Protein Conformation,
Denaturation & Renaturation
Structural changes of protein by

monitoring the fluorescence changes of a
bright and stable visible-range fluorophore
Conformational Changes in
a Saccharide Transporter
Denaturing Agents
1) Physical agents: Heat, surface action, ultraviolet light,
ultrasound, high pressure etc.
2) Chemical agents: Acids, alkalis, heavy metal salts,

urea, ethanol, guanine, detergents etc.
• Urea and guanidine probably interfere with the

hydrogen bonds between peptide linkages.
• Acids and alkalis probably attack directly the

hydrogen bonds in the secondary and tertiary
structure of proteins.
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Biological Alterations in
Denatured Proteins
• The digestability of certain denatured proteins by
proteolytic enzymes is increased.
• Enzymatic or hormonal activity is usually destroyed by
denaturation.
• The antigenic or antibody functions of proteins are
frequently altered.
• If the denaturation is severe, the protein molecules
become insoluble and precipitation results as well as the
changes in the properties of the proteins are permanent
and irreverible.
• In case of mild denaturation, there is reversible
denaturation leading to the slight changes in the
properties of the protein which can be restored to the
native state after suitable treatment.
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Lecture Summary
• Proteins consist of polypeptides folded into specific three-
dimensional configurations. In describing the three-dimensional
structure of proteins it is customary to consider four levels of
organization.
• Primary structure is the linear sequence of amino acids in a
polypeptide.
• Secondary structure refers to certain repeating conformational
pattern; namely helices, pleated sheets, reverse turns (bends)
• Tertiary structure refers to the overall polypeptide conformation.
• Quaternary structure refers to the spatial relationships between
subunits in protein that consist of two or more polypeptides
(multimeric proteins).
• Many proteins, after being unfolded artificially (denatured) can refold
spontaneously to their original (native) conformation when incubated
at an appropriate temperature, pH and ionic strength.
Classification & Function of Protein –
what makes it important
OBJECTIVES
• Know the classification of proteins and type of
functions of protein
• Give examples of some specific protein and their
functions
• Name example of some biologically active oligopeptides
• Explain the mechanism by which hemoglobin and
myoglobin reversibly bind O2.

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Classification of Proteins
Base on their chemical composition, proteins can be classified as:
• Simple Proteins
• Conjugated Proteins
• Derived Proteins

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Functions of Protein
• Our interest and curiosity in proteins stems directly from

their physiological importance and the complexity of their
functions.
• The role proteins play can be grouped into three types:

– Structural function;
– Physiological Regulatory function;
– Energy & Nutritional function.
BME2106 - Introduction to Cellular and Biomolecular Engineering BME, City University of Hong Kong 30
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Structural Proteins
• Present in all body tissue such as muscle, skin, internal

organs and cellular membranes.
• They owe their structural quality primarily to their fibrous

nature. Normally they are systematically arranged
and show strong mechanical strength.
• Both coiled form and sheet form of structure are found

in these proteins.
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Examples of Structural Proteins
Silk
• a stretched form of keratin (角疍白, 幾丁質)
which has antiparallel b-pleated sheet
structure, called b keratin.
• relatively inextensible because the side
groups of the amino acids mentioned above
are small and there is little steric hindrance.
Consequently, the peptide chains can pack
fairly closely.
• Structural strength of silk is due to the
multi-hydrogen bond cross linking
between -C=O and H-N- group.
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Horn, Hoof (蹄), Hair, Wool
• coiled form of keratin, known as  keratin;
• although structure pattern resembles that of silk fibroin,
they are highly extensible and very elastic;
• contains a high proportion of
cystine and disulphide bridges.

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Collagen (膠原) -- the protein of connective tissue which is
an extended helix of coiled coils.
• presence of unique amino acids:
hydroxyproline and hydroxylysine.
• no cystine present
• very few aromatic amino acids
• lot of glycine - the dominant amino acid
• chemically inert
• sponge-like structure and properties, can absorb water readily
• shrinks abruptly upon heated. Shrinkage depends on % of
hydroxproline present.
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Myosin (肌凝疍白) -- a globular protein
having  and 
forms region similar to keratin.
• A very large molecule of about 540
Kd.
• The protein has six polypeptides of
which
two are identical heavy
chains and four are
identical light chains.
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Myosin (肌凝疍白) -- a globular protein having  and 
forms region similar to keratin.
• The amino-terminal is a globular head while
the carboxyl-terminal is a -helical coiled-coil rod.
• This protein is totally absence of proline in the tail.
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Physiologically Active Proteins
• These proteins play an active role in

physiological processes.
• Within this category, it covers:

1. enzymes,
2. hormones nucleoproteins and
3. blood proteins.
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• Enzymes -- are organic catalysts which can speed the
rate of a specific organic reaction. Some enzymes are
specific for single chemical bonds; such as those of
amide, ester and phosphate. Others are even more
specific in that they act on a specific bond in a particular
substance only.
• Hormones -- small size proteins or peptides which hold

a key position in metabolic processes. They maintain
order in a complexity of biochemical reactions. Although
a great deal is known of their physiological action, little or
nothing of why they act in their specific ways.
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Nucleoproteins -- conjugates of nucleic acid and protein.
• In some case, the protein is wrapped around the nucleic acid to serve as a
kind of protective coat.
• In the case of the viruses, the protein also gives shape and possible triggers
off the injection of the nucleic acid into the host organism.
• There are two groups of protein commonly associated with the nucleic
acids, namely the protamines (精朊) and the histones (組疍白).
• The protamines which contain high level of arginine are found only in
sperm cells.
• The histones which are comparatively large and complex are found in
association with the DNA of the chromosomes in somatic cells. Histones
are either arginine-rich or lysine-rich. Hence the histones are also basic in
nature. They play an important part in the regulation of gene activity.
• They can also stabilize the associated nucleic acid, such as ribosomal
protein.
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Blood proteins -- Blood is multi-functional in nature.
• Its amount is equivalent to 7% of body weight and in which, different kinds
of blood protein.
• When blood proteins are concerned, they show extreme diversity of
character and a very dynamic equilibrium properties.
• Lifetime of blood protein is commonly no more than a matter of hours or
days because blood stream is the site of intense metabolic activity and the
shift of population is high.
• Blood proteins are consist of different entities: Albumin, Globulins (, ,
), Enzymes, hormones etc.
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Haemoglobin (Hb):
• Found in red blood cell. It is an oligomeric conjugated protein with
four peptide chains joined by non-covalent bonds (tetramer).
• There are several kinds of haemoglobin found in humans (A1, A2
and Fetal). They vary in the primary structure of their subunits.
• Each haemoglobin subunits contains a pocket for the heme that
provides the nonpolar environment needed by the heme-iron in to
order to bind O2 reversibly.
• The heme-iron bonded to histidine residue on one side of the heme
plane. At oxygenation, iron liganded to O2 on the opposite side of
the heme. Heme-iron remains in Fe++ all the time.
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CH 2
H CH 3
C Structure of Heme. Heme
H3C
CH 2 consists of a porphyrin ring
N N
(composed of four pyrrofes)
HC Fe CH with an Fe-II in the center.
N N
H3C CH 3
C
H
COO - COO -
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• Haemoglobin is the main O -carrying protein. It accounts for 98%
2
of O2 transport. It carries oxygen from the lungs to the tissues and

carbon dioxide from the tissues to the lungs.
• The oxygen-binding curve for haemoglobin is sigmoidal in shape. It
has a high affinity for O2 at the relatively high oxygen tension in the
lung and is able to release it at the relatively low oxygen tensions in
the tissue because myoglobin can bind stronger.
• At high O2 tension, Hb binds O2 thousand times tighter than
mammalian myoglobin (Mb). But Mb requires a much lower [O2] for
50% saturation than Hb.
• Haemoglobin is also the major factor in the control of blood pH at
7.3-7.5.
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Nutrient Proteins
• Proteins are required nutritionally because most non-plant
organisms (animals) are unable to elaborate their own protein
requirements from elemental nitrogen.
• They need an external supply of proteins, as well as fat and
carbohydrates, as raw material for their own biological systems.
These systems, however, have a curious inability to produce certain
amino acids, known as essential amino acids which must be
present in the diet.
• In the case of human nutrition, the essential amino acids are
valine, leucine, isoleucine, threonine, lysine,methionine,
phenylalanine and tryptophan. Their effect on the growth of young
animals is shown in the figure on the right.
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How do we detect these proteins?
quantitative enzyme-linked immunosorbent assays (ELISA)
Western blotting
Immunostaining
How do we detect these proteins via
bioengineering approaches?
2. Capitalising on the odd
Harnessing differences for management

strategies
Sorting for detecting and disease monitoring - isolating the odd ones – by biomarker expression (e.g.
CD44, CK, EpCAM)

strategies
Sorting for detecting and disease monitoring - isolating the odd ones – by size (varied distribution
along a curvilinear channel under inertial force)
Target cell outlet Waste outlet
Khoo et al. 2015 PLOS One

Clearbridge

strategies
Sorting for detecting and disease monitoring - isolating the odd ones – by size (differences in
migration patterns)

strategies
https://www.pnas.org/content/101/29/10501.short

strategies
https://pubs.rsc.org/en/content/articlehtml/2015/lc/c4lc01058b
Challenges in detection of ‘the odd ones’
• Have to be carefully studied with rigorous computation,

which is imperative to distinguish pre‐existing genetic
alterations from amplification errors.
• Multiple independent samples. biasness and errors,
especially in highly heterogeneous samples,
• Since it is technically impossible to eradicate sampling
noise due to the low amount of sample material, additional
steps may be required to distinguish noise from low
prevalence signals. These may be carried out with molecular
fluorescence in situ hybridization (FISH) or unique molecular
identifiers.
• Noise structure from single cell sequencing can also be
determined to differentiate sampling noise from biological
signals.
2. Targeting ‘the odd’

strategies
Personalised targeted therapy

strategies
• Therapy targeting a mutation present in only a fraction of

tumor cells would be expected to affect only that subclone,
leading to limited clinical benefit.
• At worst, targeted therapy might have a paradoxically
stimulatory effect on the subclones lacking the relevant
mutation.
• Important to enumerate the extent of clonal heterogeneity in
patients being evaluated for targeted therapy and to interpret
the results of subsequent therapy in light of such genetic
heterogeneity.

strategies
• Effective targeted therapy will require either drug

combinations targeting distinct subclones or,
• Deployment of targeted therapies only in patients for whom
the drug target is entirely clonal.
• Monitoring over time - patient risk factors, patient response,
and patient adherence ( adaptive treatment strategy concept
used in substance abuse)

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BME2106 - Introduction to

Cellular and Biomolecular

Dr. Bee Luan KHOO

Department of Biomedical Engineering

Where does the amino acid come from?

• The three most abundant regular secondary

4 of these make 1 of this Termination of helical structure:

Note: H bonds that hold the sheet together are

A β-turn allows the polypeptide

• The other interactions are

The quaternary structure of a globular protein composed of two

• Many proteins, after being unfolded artificially

Where T is the temperature in Kelvin.

Structural changes of protein by

2) Chemical agents: Acids, alkalis, heavy metal salts,

• Urea and guanidine probably interfere with the

• Acids and alkalis probably attack directly the

HYC\Molecules&Cells\ 2017/1/26 2828

Base on their chemical composition, proteins can be classified as:

HYC\Molecules&Cells\ 2017/1/26 2929

• Our interest and curiosity in proteins stems directly from

• The role proteins play can be grouped into three types:

• Present in all body tissue such as muscle, skin, internal

• They owe their structural quality primarily to their fibrous

• Both coiled form and sheet form of structure are found

HYC\Molecules&Cells\ 2017/1/26 3319

• These proteins play an active role in

• Within this category, it covers:

• Hormones -- small size proteins or peptides which hold

of O2 transport. It carries oxygen from the lungs to the tissues and

Harnessing differences for management

Harnessing differences for management

Target cell outlet Waste outlet

Khoo et al. 2015 PLOS One

Harnessing differences for management

Harnessing differences for management

Harnessing differences for management

• Have to be carefully studied with rigorous computation,

Harnessing differences for management

Harnessing differences for management

• Therapy targeting a mutation present in only a fraction of

Harnessing differences for management

• Effective targeted therapy will require either drug

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