Introduction to

macromolecules: Proteins
Dr. Maduka de Lanerolle-Dias
Senior Lecturer
Dept. of Biochemistry and Molecular Biology
Learning outcomes
Discuss Discuss protein turnover including the fact that proteins are not primarily a
protein store

Outline Outline the basic structure of amino acids that are found in proteins including
the broad categories that they can be classified into depending on R group
properties and those that are not found in proteins

Discuss Discuss amino acids as weak acids or bases with a potential role in buffering

Apply Apply an understanding of pKa to determining the charge of an amino acid in

physiological pH

Explain Explain how the R group and the peptide bond structure confers different
properties to proteins which enable different functions

Discuss Discuss the importance of the primary sequence in determination of structure

Discuss Discuss the structure/function relationship in small and lager peptides

Discuss Discuss the 4 levels of organization of proteins in relation to functional

requirements with examples

Compare Compare the favoured structures in functional versus structural proteins

Explain Explain the requirement of a microenvironment to synthesise secretory

proteins and describe the mechanism for protein folding following translation

Explain Explain how posttranslational modifications improve function.

Describe Describe conjugated proteins in relation to function.

Describe Describe the plasma protein types and function and their normal
electrophoretic pattern

Explain Explain the requirement for unused proteins, wrongly synthesized and folded
proteins to be degraded, in a microenvironment, to aa which enter the pool.

Outline Outline at which points problems that lead to disease states can occur
PROTEIN TURN OVER
• Proteins are constantly being broken down
and re-built
• Therefore, proteins transition between
various states/ structures
• This is the dynamic nature of proteins
• Protein turnover : the continual renewal or
replacement of protein.
• Defined by the balance between protein
synthesis and protein degradation.

! Unlike fats and

carbohydrates, amino
acids are not stored by the
body

! Any amino acids in

excess of the biosynthetic
needs of the cell are
rapidly degraded.
 Nitrogen Balance
•A balance between the amount of Nitrogen intake (in
the form of dietary protein) and amount of Nitrogen
lost/excreted (in the form of urea, uric acid, creatinine
and small amount of amino acids. By an individual.
•N equilibrium : intake = output
Nitrogen Nitrogen
Consumption excretion

Positive Nitrogen balance

•Intake > loss
•Anabolism
•Growth, pregnancy, recovery from
illness
•Hormones that lead positive nitrogen
balance
• Growth hormone, insulin, Androgens

Nitrogen
Consumption
Nitrogen
excretion
Negative Nitrogen Balance
•Loss > intake
•Catabolism
•Chronic/acute illness, protein deficiency starvation
•Hormones that lead to negative nitrogen balance
• Corticosteroids, T4
Nitrogen
excretion

Nitrogen
Consumption
Amino acid structure
Amino acids are the building blocks of
proteins and they are organic molecules

Formation of esters
Acylation Formation of acid anhydrides
Amidation Formation of amides
Reduction of carboxyl group

OH & SH
groups

oxidation &
esterification

The 20 aa Can be categorized

according to the R group

Positively charged,
Non-polar,
R groups
aliphatic R groups
(Hydrophobic)

Negatively
charged, R groups

Polar, Unchanged R
groups
Non-polar,
aromatic R groups
(Hydrophobic)
Amino acids can
also be classified
according to the
dietary
requirement

Apart from being the building

blocks of proteins, amino acids
(and their derivatives) have
other functions
–Nerve transmission
–hormone
Messengers (hormones,
releasing factors)

–ofPrecursors for the biosynthesis

moleules (heme, pyrimidines,
purines, porphyrins, urea)

–Forming parts of coenzymes

Zwitterions and Amino Acids
•Zwitterion : a molecule with at least one functional
group with a positive charge, and at least one
functional group with a negative electrical charge.
•Net charge of the entire molecule is :
•zero.
•The (neutral) zwitterion is the usual form amino acids
exist in solution.

•The unionized form does not exist in cells.

•Depending on the pH, there are two other forms, an
anion and a cation:

•Predominant form depends on the R group & pH of

medium
The isoelectric point (pI) / (IpH)

• the pH at which a particular molecule

carries no net electrical charge.
• Predominately zwitter ion
• Equal but very small amounts of
cations and anions
• Solubility is lowest
pI =pk1 + pk2
2

Buffering action

•Amino acids can act as both an acid AND a base -->

buffering action
(it can react with added acids and bases to keep
the pH nearly constant.)
•Maximum buffering actions when pH=pKa
•pKa tells you what the pH needs to be in order for a
chemical species to donate or accept a proton

pH - a measure of the concentration of hydrogen ions in

an aqueous solution.
pKa - describes the point where the acid is 50%
dissociated (i.e. deprotonated).
•Imidazole group of Histidine (pKa ≈7) is important in
the buffering action of Hemoglobin (Hb)- pK of amino
acid is slightly altered when it is in the protein
(protein in Hb, “globin” is rich in His)
Below a pH of 6.0,
histidine act as a
proton acceptor Above a pH of 6.0,
(The imidazole ring is histidine acts as a
mostly protonated) proton donor

The type of amino acid and its properties as well as the

sequence of amino acids in a chain will determine the structure
as well as the function of a protein molecule.

Side chains contributing to charge

R group Conj. acid Conj. base pKa

Non-α COO- -COOH RCOO-
4.0 R
(Asp, Glu)
R
Imidazolium HN NH+ HN N 6.0
His
Non-α -Amino R-NH3+ R-NH2 10.5
(Lys)
Phenolic OH R- -OH R- -O- 10.1
Tyr
Guanidinum - C=NH+ -C=N 12.5
(Arg)
-
Sulphydryl (Cys) R-SH R-S 8.3

Slide imported from Lecture by Prof. Pulani Lanerolle, on “Introduction to Macro molecules : Proteins”
Primary structure – unique
Aa sequence is specified
Forms H bonds
between –CO and -NH Three-dimensional Several protein
by the sequence of arrangement of chains /subunits in
nucleotides in the coding groups polypeptide chain in a closely packed
region of the DNA space. arrangement.

• Amino acids are joined covalently by

peptide bonds and form peptides
and proteins
• The peptide bond is rigid and
planer, C,O,N,H are on the same
plane
 Primary
structure

The peptide bond has a partial double-bond character,

Therefore rigid
prevents free rotation around the bond between the
carbonyl carbon and the nitrogen of the peptide bond.
bonds between the α-carbons and the α-amino or α-
carboxyl groups can be freely rotated

Intra-chain H
bonding

Secondary
structure

Inter-chain H bonding
Different polypeptide chains
Or
Different parts of same chain
 Stabilized by:
Outside polar hydrophilic hydrogen and ionic bond interactions

Internal hydrophobic interactions between nonpolar amino acid

side chains

Tertiary
structure

Posttranslational covalent bonds in

the tertiary structure may be formed
with prosthetic groups.

Based upon their tertiary structure,

proteins are divided into
1. Globular proteins
2. Fibrous proteins

Fibrous proteins (i.e α-keratin):

Elongated rope-like structures that are
strong and hydrophobic.

Globular proteins (i.e plasma

proteins, immunoglobulins):
More spherical and hydrophilic. immunoglobulin

Plasma retinol
binding protein
α-keratin
 Globular vs Fibrous protein
Globular Protein Fibrous Protein
Shape Spherical in shape elongated strand-like structures (rods
or wires.)
Solubility Soluble in water (forms colloids), insoluble in water, weak acids and
acids and bases weak bases but soluble in strong
acids and alkalis
Example Hemoglobin , myoglobin, α-keratin, collagen and elastin
lysozyme, ribonuclease
Bonds held together by weak peptide chains are bound together by
intermolecular hydrogen bonds. strong
intermolecular hydrogen bonds
Function Functional proteins Structural

Lysozyme

Pleated
sheet

α-helix

Slide imported from Lecture by Prof. Pulani Lanerolle, on “Introduction to Macro molecules : Proteins”
 Structure of myoglobin
Haem
Fe 2+

O2 or H2O
F
E

Distal His
His (E7)
Proximal
His (F8)

Slide imported from Lecture by Prof. Pulani Lanerolle, on “Introduction to Macro molecules : Proteins”

Immunoglobulin (antibody) structure

Antigen binding
domain

Intrachain
disulphide bonds

Slide imported from Lecture by Prof. Pulani Lanerolle, on “Introduction to Macro molecules : Proteins”
 Levels of organization of collagen fibrils

Secondary

Primary

Slide imported from Lecture by Prof. Pulani Lanerolle, on “Introduction to Macro molecules : Proteins”

• Each subunit has its own primary, secondary, and tertiary structure.
• Held together by hydrogen bonds and van der Waals forces between nonpolar side
chains.
• need to be arranged in a specific way for proper function
• Alteration results in changes in activity

Quaternary
structure
Protein (subunits) Function
Alcohol dehydrogenase (4) Enzymatic reaction in fermentation

Aldolase (4) Enzymatic reaction in glycolysis

Fumarase (4) Enzymatic reaction in citric acid cycle

Hemoglobin (4) Oxygen transport in blood

Insulin (2) Hormone that regulates metabolism

of glucose
Classification of proteins
according to shape
Fibrous proteins Globular Proteins:

• Polypeptide chains • Polypeptide chains,

folded in to filaments/ folded in to compact,
sheets spherical/ globular
• Water insoluble shape
• Regular IIry structure, • Water soluble
no defined IIIry st. • Secondary, tertiary (&
• Structural - quaternary) structure
Protective/supportive • Functional -
role (Collagen, Keratin, Haemoglobin, plasma
elastin) proteins such as
• Connect connective albumin, globulins,
tissue, tendons, bones enzymes, hormones
and muscle fibers
 Conjugated proteins
A simple protein + non-protein component (= prosthetic group)

Conjugated protein Protein part Prosthetic group

Chromoprotein Protein Coloured compound
(Haemoglobin) (Globin) (Heam)

Glycoproteins & proteoglycans Protein Carbohydrate

(Mucin of saliva)
Lipoprotein Protein Lipid

(cell membrane, plasma lipoprotein)

Nucleoprotein (chromosome, ribosome) Protein nucleic acid
Phosphoprotein Protein phosphate

(casein linked via Ser, Thr )

Protein folding

Misfolding

Proteins must fold to provide their functional/

structural support
incorrect folding → aggregation → formation
of toxic species
Misfolding may occur:
Spontaneously
or by a mutation of a particular gene
or by other stimuli
Misfolding is prevented by guided folding -
molecular chaperones
 Protein folding with
chaperones
- Molecular chaperones are proteins
that, facilitate the folding of other
proteins.
- Reversible

two families of chaperones

1. Heat shock proteins
2. Chaperonins

Heat shock proteins (Hsps)

A large family of molecular

chaperones
Numerous stressors, including
hyperthermia and hypoxia, can
induce the expression of Hsps,
which, in turn, interact with client
proteins and co-chaperones to
regulate cell growth and survival.
REVIEW article: Front. Neurosci., 12 November
2018 | https://doi.org/10.3389/fnins.2018.00821
Chaperonins
large, double-ring oligomeric proteins that act as containers for the folding of
other protein subunits.
HSP60, also known as chaperonins (Cpn), is a family of heat shock proteins
not all chaperonins are heat shock proteins

Ranson, N. A., White, H. E., & Saibil, H. R. (1998). Chaperonins. The Biochemical journal, 333 ( Pt 2)(Pt 2), 233–242.
https://doi.org/10.1042/bj3330233

age-related decline in proteostasis capacity allows the

manifestation of various protein-aggregation
diseases, including Alzheimer's disease and
Parkinson's disease.
 Formation of amyloid fibrils

Slide imported from Lecture by Prof. Pulani Lanerolle, on “Introduction to Macro molecules : Proteins”

Natural protein Infectious protein

+ Pleated
sheet
α- helix

H bonds

Model for change in conformation of prion protein

 • Total plasma or serum
Plasma vs Serum proteins 70-80 g/L
•Plasma protein fractions:
Albumin (main ) – 35-
50 g/L
Globulins
−
α1 globulin
−
α2 globulin
−
β globulin
−
γ globulin
(immunoglobin)

https://www.youtube.com/watch?v=5Ek5s
-LfBXA
 Separation of plasma
proteins
Plasma proteins can be separated into albumin, globulins (alpha,
beta, gamma), fibrinogen by, electrophoresis

• Gel electrophoresis

• Capillary electrophoresis – greater resolution (separation of beta

1&2
components), no staining
required

Electrophoretic pattern of normal serum

globulins
(immunoglobulins)

origin
+ -

Plasma - contains fibrinogen peak between β and γ

 Report

Electrophoretogram of serum

Normal

+ - origin

Increased γ-
globulin
+ -

Sharp γ - band
paraproteinaemi
+ - a