Section II: AMINO ACIDS, PEPTIDES & PROTEINS

Receptor binding domain

❑ Amino acids – structures & properties.

❑ Peptides
❑ Proteins –
o Organization: primary, secondary, tertiary &
quaternary structures.
Single protomer of
2019-nCoV S

o Dynamics: Protein folding

Science 367: 1260-126 - 2020

 We know how covid 19 use specialized
proteins to enter cells

CDC
Protein-Protein
interactions

J Adv Res 24:91-98,

2020 Life Cycle of Covid-19 (SARS-Cov2)

https://psychscenehub.com/psychinsights/covid-19-and-the-brain-pathogenesis-and-neuropsychiatric-manifestations-of-sars-cov-2-cns-involvement/
 Central theme for vaccinaton – how protein-
protein interact

e.g., Pfizer/BioNTech (EU); Moderna (US)

e.g., 'Sputnik V‘, RU Covid 19

e.g., Sinovac Biotech, CN virus
e.g., Serum institute, Ind

Trimeric Spike protein

Immunity 52(4):583-589, 2020 07Dec 2020

COVID-19 vaccine tracker (shinyapps.io)
Receptor binding domain
(Omicron has many mutations)

Challenges
ACE2

❑ How to design the correct target with

the right conformation Human cell

❑ How to produce enough

What do proteins do in a cell?
Learning: proteins are “workhorse” of cell processes

Alberts et al

Examples:
1. Structural functions – act as scaffold of a cell.
2. Enzymes – e.g., amylase/digestive enzymes.
3. Transcription factors -participate in reading genetic information/making RNA.
4. Translation factors- participate in making proteins using genetic information.
5. Hormones – e.g., insulin.
6. Intracellular signaling – eg. kinases.
Therapeutic Proteins

Gold ~ $50-100 (USD)/g

20th Dec 2021– ~$57 USD

3rd Aug 2023 - ~ $61 USD

Erythropoietins - >$1,000,000 (USD)/ g!

 Many proteins are best selling drugs

Produced by Biotechnology Produced Chemically

“protein drugs”
Biomolecules have complex molecular structures

Erythropoietin
Interleukin (cancer,
(anemia, kidney
immune disease)
disorder)

Tissue plasminogen
Insulin hexamer activator
(Diabetes M) (acute myocardiac
infarction)
Why study the structures of proteins?
Learning: structure of proteins correlates with functions of the proteins

❑ Changing structure of the protein can change the function.

Therefore, if we understand how protein structures are organized from simple

arrangements of amino acids (building blocks), we can use this information for
drug design and figure how they work in silico.

Fold

From sequence to 3D
 Critical ASsessment of Techniques for Protein Structure
Prediction (http://predictioncenter.org/)
❑ 15th Community Wide Experiment (2022)
Started in 1994 (CASP15: May-August 2022)

❑ Modelling is getting more accurate

Google DeepMind AlphaFold technology

outperformed 100 other teams. Using AI

o Predicting folding accurately (>90%) of

segments (not entire proteins)

o Including Orf8 (Covid 19)

o De Novo prediction of structures for designing

Highly accurate protein structure prediction with AlphaFold.
Nature volume 596, pages583–589 (2021) therapeutics
Each “key and lock” (protein-protein interaction) sets
have different structures
Learning: need more accurate/faster ways to determine structures

❑ Similar to the concept of “Lock-Key” (but more flexible).

Only those proteins that “fit” the lock will work!

❑ Human genome codes for ~ 20,000 - 25,000 different proteins

❑ Many more protein structures due to alternative splicing & post-

translational modifications
How different proteins adopt different conformations
Learning: same compositions of amino acids but different arrangements – may
give rise to different conformations of the proteins

❑ Secret lies in the way the protein is assembled from individual building blocks
– interactions and properties of the building blocks (amino acids)
NH2 NH2

COOH
Amino acids
Amino acids are building blocks of Proteins
Learning: proteins are polymers of amino acids

R O R O
+ - Amino acid
H2 N CH C OH H3 N CH C O

pH7: 0 net charge when R is H (as in glycine)

Zwitterion

Amino acid 1 Amino acid 2 Amino acid 3 Amino acid 4

Polypeptide chain

Peptide bond
AMINO ACIDS – Building blocks of proteins
Learning: all “standard” amino acids are -amino acids. R group can be
different. Except for proline, all amino acids have free NH2 and COOH group

❑ Chiral center (because of tetrahedral bonds of carbon)

Protein groups are non super-imposable

 -carbon of most amino acids are chiral centers
(except glycine)

❑ Thus, amino acids have stereoisomers.

In cells: predominantly L-amino acids exist.
Very few D-Amino acids (largely seen in frog skin as a result of evolution)
- L-isomer recognising enzymes in humans will NOT recognise D-isomers

R
H2 N COOH
H R

H2 N H

COOH
H COOH

NH2

Fingers/tumb to illustrate asymmetry.

Formation of peptides & proteins
Learning: COOH /NH2 groups of two amino acids condense resulting in
the loss of a water molecule to form a covalent amide bond.
20 standard Amino Acids
Learning: physicochemical properties of the R-group can be used for
classification

R O

H2 N CH C OH

AMINO ACIDS

NON-POLAR POLAR

Aliphatic Aromatic Acidic Basic Uncharged

(The comparisons are made between each amino acid)

Non-Polar Amino Acids

Dark chocolate – increase serotonin

**Just need to remember the key groups on each protein
Structure and PKa given on exams

metabolism
Serotonin
(mood – Prozac inhibit uptake of
serotonin in neuron)
Polar uncharged side group

Neurotransmitter

Refer to picture for more info (Tele

Reminders)
**Electron delocalisation: Double bond can
simultaneously occur on CO and CNH2

Not a good nucleophile (NH2)

and do not accept H+ as
readily as lysine
POLAR acidic side group

Watch lecture

❑ Important “excitatory” neurotransmitters

(epilepsy) – chinese restaurant syndrome

❑ “Umami taste” – binding to taste receptors

POLAR basic side group

Watch lecture

Animal feed

+ +

Need to feed lysine

Amino acids grouped into ‘essential’ & ‘non-essential’
Learning: another way of classifying amino acids based on metabolism

❑ Starvation – in order to generate energy:

Carbohydrates  Fats  Proteins [amino acids are not stored]

Obtain from diet

ESSENTIAL NON-ESSENTIAL

❑ We cannot make all amino acids. Arginine Alanine

Histidine Asparagine
Plants/microbes make all these. Isoleucine Aspartate (Aspartic acid)
Leucine Cysteine
Lysine Glutamate (Glutamic acid)
❑ ‘Essential’: does not biosynthesize Methionine Glutamine
Phenylalanine Glycine
Threonine Proline
Tryptophan Serine
Valine Tyrosine
Amino acids: different frequencies found in proteins
Learning: different proteins have different amounts of a particular amino acid
Some amino acids more frequent than other amino acids in proteins
Amino acids have 3D structure
Learning: although they share common chemical backbone, the overall shape is
different

Glycine Alanine Phenylalanine

❑ Proline – cyclic amino acid. Conformational constraints

❑ Except for proline all other amino acids have a free NH2 group

Illustration – ..\..\rasmol\pdb-amino acids\asparagine.pdb

Acid-Base properties of amino acids
Learning: different R groups affect ionization of amino acids

❑ Amphoteric molecules (have characteristics of acid & base)

❑ At least 2 pKas, and 3 if R group has dissociable protons (e.g., aspartate, lysine)
Titration of Glycine

pK2 9.6

No net charge
pI = 5.9
pK1 2.3

pI = 9.6 + 2.3
❑ pI = pKa/2 ; pKa of groups that will lose/gain 2
charge of the neutral species.
= 5.9
Titration of Glutamic acid
**COOH above disassociates before COOH below bc H3N+
attracts electrons, thus making it easier for H ions (positive) to
escape. The bottom COOH has a stable bond thus is less likely to
disassociate first

pK3 9.7

pK2 4.2

pK1 2.2 pI = 4.2 + 2.2

2
= 3.2
Titration of lysine

❑ Note: isoelectric point is

more basic than glutamate

pK3 10.8
When you have 1 pKa, it CANNOT have a pI because the
graph will reach an asymptote (eventual plateau)

pK2 9.2

pI = 9.2 + 10.8

pK1 2.2 2
=10
Other natural amino acids found in some proteins
Learning: beside the usual amino acids, there are 2 others

❑ Example of an important intracellular enzyme containing selenocysteine:

thioredoxin reductases

Cysteine Selenocysteine

❑ Special tRNA used for biosynthesis.

❑ Encoded by a UGA codon (normally a stop codon).
*Found in human
Another rare natural amino acids found in some proteins

❑ Methylpyrroline-carboxylate group linked by amide bond to lysine at the 

amino group.

 
 Lysine
 
Pyrroline

Pyrrolysine

Methyl
Carboxyl
Lysine

❑ Special tRNA used for biosynthesis.

❑ Encoded by a UAG codon (normally a stop codon).
* Not reported in human
 Modified amino acids found in some proteins
Learning: some amino acids in certain proteins can be chemically modified for
special functions

Muscle proteins Histone proteins Collagen

❑ Formed as a result of post-translational modifications.

❑ Specific for only some proteins.
❑ Perform important functions in proteins.
Other modified amino acids in some proteins





Glutamic acid

-Carboxyglutamic acid Protein

❑ Defective carboxylation of the glutamate residues in prothrombin (a clotting

protein) can lead to haemorrhage.

❑ Prothrombin must bind calcium before activation to thrombin

❑ Normal prothrombin contains several residues of -carboxyglutamate

 Other modified amino acids in some proteins

Phosphoserine/Phosphothreonine/Phosphotyrosine

❑ The most common modified amino acids

❑ Phosphate groups add negative charges to protein

❑ Present in abundance in casein (milk protein) –

binds calcium due to –ve charges (phosphoserine).

❑ Involves in cell signaling and control of enzyme

activity. Reversible phosphorylation. Some growth
factor receptors “switch-on-off” by
phosphorylation.
Proteins involved in cell signalling
Learning: reversible post-translational modification of amino acids on
proteins has unique functions

❑ Cells convey signals from external to internal by exploiting

reversible changes in some amino acids – e.g., phosphorylation.

PO4- added to tyrosine on receptors

**Dont memorise

Cell signalling involves relay of complex information

❑ Involves relaying information

from protein to protein

* For information only

Other post-translational modifications

Acetylation (histones)

Myristoylation

Palmitoylation

Prenylation Adenylylation
ADP-ribosylated histidine
Derivatives of amino acids Not found in proteins
Learning: some amino acids are metabolized to form other compounds

Glutamic acid Histidine Tryptophan

Metabolism
 Peptides & Proteins

Short polymers of amino acids

❑ Each unit is called a residue

❑ 2 residues - dipeptide
❑ 3 residues - tripeptide
❑ 12-20 residues - oligopeptide
❑ many residues – Polypeptide/Proteins
Peptides/polypeptides/proteins are formed by
dehydration of amino acids

Condensation
Example of a tetrapeptide

What is this?

Leucine enkephalin
Nutrasweet – “sugar free sweetener”
Learning: peptides can be synthesized to have special functions

* Modified C terminus
 Biologically active peptides
Learning: short peptides have special functions through receptors on cells

❑ Oxytocin 9aa Contraction of uterus

(Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-amide)

❑ Vasopressin 9aa Antidiuretic

(Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-amide)

❑ Beefy meaty peptide (“delicious peptide”) 8aa Food beefy flavouring

(Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala)

❑ Substance P 11 aa pain transmission

(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-amide)

❑ Met-Enkephalin 5aa pain (morphine mimics)

(Tyr-Gly-Gly-Phe-Met)

Can be made synthetically (~20-60 amino acid long) in the

laboratory.
 Natural anti-microbial peptides

Naturally
occurring Microorganism
Frog species
antimicrobial targeted
peptide
Multidrug-resistant Alyteserin-1c (GLKEIFKAGLGSLVGIAAHVAS-NH2)
Midwife toad
Alyteserin-1c Acinetobacter
Alytes obstetricans
baumannii
Extended-spectrum β- Ascaphin-8 (GFKDLLKGAA KALVKTVLF)
lactamase
Tailed frog Ascaphus Ascaphin-8
(ESBL)Klebsiella
Pseudin-2
pneumoniae
(Gly-Leu-Asn-Ala-Leu-Lys-Lys-Val-Phe-Gln-Gly-
Paradoxical Frog Antibiotic-resistant Ile-His-Glu-Ala-Ile-Lys-Leu-Ile-Asn-Asn-His-
Pseudin-2 Val-Gln)
Pseudis paradoxa Escherichia coli
Methicillin-
California red-legged Temporin A (FLPLIGRVLSGIL-NH2)
Temporin-DRa resistant Staphylococcu
frogRana draytonii
s aureus (MRSA)
Methicillin- XT-7 (GLLGPLLKIAAKVGSNLL-NH2)
Tropical clawed frog
XT-7 resistant Staphylococcu
Silurana tropicalis
s aureus (MRSA)
 Charges on Amines

Tertiary amine

Primary amine

Secondary amine Secondary amine Tertiary amine

but no charge has charge
Biological functions of proteins

Proteins are the agents of many biological function

❑ Enzymes - amylase

❑ Regulatory proteins (hormone) - Insulin

❑ Transport proteins - Hemoglobin

❑ Structural proteins - Collagen

❑ Contractile proteins - Actin, Myosin

❑ Exotic proteins - Antifreeze proteins in fish

Thermostable polymerases (used in PCR)
 Human proteins in the genome
Learning: attempt to classify proteins (predicted/validated) based on structures &
functions

PANTHER version 16 (2021)

https://doi.org/10.1093/nar/gkaa1106
Proteins: large and small
Learning: proteins are generally too complex (folding) to be made chemically.
❑ Usual to produce these by biotechnology
❑ Some proteins form large subunits
 Protein assembly

22 (heteromeric)
- 141 amino acids
 - 146 amino acids
12 (homomeric)
Each chain – 468 amino acid
Rasmol.
Proteins have different overall shapes (conformations)
Learning: proteins can be grouped based on solubility & shape

❑ Fibrous proteins
❑ Globular proteins
❑ Membrane proteins
 Proteins in different locations

Rbc ‘ghost’

Hypotonic medium

SOLUBILIZE WITH DETERGENT

RED BLOOD CELLS contain many types of proteins

Load samples
Cathode (-)

Buffer
Gel Anode (+)
(Polyacrylamide[
PAGE])
DECREASE IN SIZE
Buffer
Steps in polyacrylamide gel electrophoresis
Casting gel Ready for sample

Loading samples Gel ready for electrophoresis

Run gel
(electrophoresis)
 Shape of RBC is maintained by
many proteins arranged in 3-D

ELECTRON MICROGRAPH:
proteins arrangements in
the inner membrane of RBC

Modified Karp
Cell membranes contain proteins of different topologies
Learning: proteins can be grouped based on location

INTEGRAL PROTEINS

Receptors, transporters PERIPHERAL PROTEINS:

Proteins that are associated
❑ Intracellular proteins with the membrane

❑ Extracellular proteins

LIPID ANCHORED PROTEINS:

Proteins linked via lipids to the
membrane

Modified Karp
Globular shaped Proteins
Learning: proteins can be grouped based on shape

❑ Soluble in aqueous medium. Diffuse readily.

❑ Tightly folded into compact globular shape with hydrophobic residues inside
and hydrophilic residues on surface.

❑ Structurally complex - contains several types of 20 structures.

e.g., enzymes, transport proteins, antibodies.

 Fibrous shaped Proteins
Learning: proteins can be grouped based on shape

❑ Insoluble in aqueous medium

❑ Extended structures with hydrophobic residues on the outside

❑ Consist largely of one type of 20 structure

❑ Structural or protective role

e.g., a-keratin, collagen, fibroin (silk)

REPEATIVE UNITS of :
Gly-X-Pro,
or
Gly-X-HyPro,
Where X is any amino acid
Some Proteins are modified
Learning: some proteins are post-translationally modified

❑ Simple protein contains only polypeptide chain (sometimes with modified

amino acids).
❑ Some proteins have other modifications - Conjugated proteins
(polypeptide chain + prosthetic group – organic or inorganic moiety).
❑ Classification based on nature of prosthetic group

Proteolipids

Covalent-linked
How/Why proteins adopt specific structures
 Structural Organization of Proteins
Learning: shape can be described at 4 levels of organization

Primary Secondary Tertiary Quartenary

linear arrangements local folds global folding aggregation of
global folds

Modified Nelson & Cox

Primary Sequences
❑ Refers to linear sequence of amino acids
number - amino acid composition
sequence - amino acid sequence

❑ Also location of S-S bond(s), if any.

Insulin – 2 linear sequences (chains

A & B) linked by disulphide bonds.

❑ *do not show 3D or local structures

Glial Cell-line Derived Neurotrophic Factor
(GDNF) – different ways to visualize a protein

Cartoon
representation

Ball-stick
Wire-frame

Animation.
Proteins have specific conformations for function

Linear PRIMARY sequence of modified GDNF containing C-terminal 134 amino acids.
MSPDKQAAALPRRERNRQAAAASPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCEAAETMYDKIL
KNLSRSRRLTSDKVGQACCRPVAFDDDLSFLDDSLVYHILRKHSAKRCGCI

Secondary structures

-Turns

-Sheets

Loops

-Helix
Forces contributing to shape of proteins
Learning: Various non-covalent forces stabilize protein structure

❑ H-bonds
Hydrophobic residues buried inside molecule
(shielded from the environment) ❑ Hydrophobic interactions
❑ Electrostatic interactions (ionic)
❑ Van der Waals interactions
Hydrophobic interactions of different
residues hold molecule in shape

❑ The R-group of side chains

contributes to these forces.

Hydrophilic interactions
with water – H bonds
Geometry of peptide backbone
Learning: Peptide bond in its usual trans conformation of carbonyl O/amide H
Peptide bond characteristics
Learning: delocalized bonds result in shorter
bond length resulting in a rigid bond – amide
bond has partial double-bond character.
Amide bond properties
Learning: due to rigid configuration, C-C-N-C rotates as a plane – coplanar
relationship of atoms in amide group

❑ Dipole generated by resonance structure-slightly polar

❑ Non-chemically reactive – N cannot react as a nucleophile (electron donor)
Amide plane
2 degree of rotation -  and  angles

Tetrahedral bonds of -Carbon

Fully extended dipeptide

(proteins are not fully extended structures)
 Steric hindrance limits the number of
conformations about -Carbon – amide plane
Learning: most combinations of  &  angles are sterically forbidden.
 Structures are constrained by the amide planes
Learning: by calculating  &  angles for every residue it is possible to
produce a conformational plot – Ramachandran plot

N- terminus
C- terminus

Bond is not free

to rotate

Modified Nelson & Cox

Sterically reasonable values of the angles  & 

Dots in purple indicate actual angles

measured for 1000 residues in eight proteins

❑ Limited number of angles are allowed

Ramachandran Plot: shaded portion are favorable angles

+180
 (deg)

-180
-180 0 +180
 (deg)
Actual angles measured & Ramachandran plot

Cytochrome C +180

(deg)
0
(deg)

-180
-180 0 +180
 (deg)

 (deg) Plastocyanin

 (deg)

 (deg)
Because of the constraints of how the amide planes
can “twist”, there MUST only be a limited number of
favorable secondary structures.
 Few types of secondary structures are stable and
occur widely in proteins -  helices and  sheets

Side view

Front view

Rasmol
Secondary Structures most prominent: -helix & -sheet

1 Conformation stabilized largely by weak interactions

2
3
❑ Rod-like structure
4 H-bonding
❑ R-groups extend outward in a helical
array
❑ Intra-H bond spacing is 4 residues apart
❑ 3.6 amino acid residues/turn
❑ Proline residues interrupt a-helix
❑ Commonly found in globular proteins

Lodish et al
 Helix and amino acids
Learning: proline & glycine are not commonly found inside alpha-helices

❑ Proline generally disrupts a-helix formation because of

the backward twist of the gp.-destabilizing kink.
❑ Hence, proline is seldom found in helices but at the
start/end of helices.

❑ Glycine is too flexible and prefer to adopt a

random coil structure.

Nature Structural Biology 9, 318 - 319 (2002)

-SHEET

❑ Extended “sheet-like” structure

❑ Intermolecular H-bonds
❑ Parallel or antiparallel -sheets
❑ Favour amino acids with compact R groups
❑ -(Gly-Ser-Gly-Ala-Gly-Ala)n- sequence motif in the silk protein, fibroin
R
N
-sheet -
antiparallel O
-SHEET can be parallel or antiparallel
Learning: depending on whether it is parallel or antiparallel, the H-bonding is
structurally different

O
H
Parallel N

Antiparallel
Other structures

Glycine Proline
2
❑ Helices/-sheets: ~50% of regular 20 structures of 3
4 1
globular proteins

❑ Others: coil or loop; reverse turns, -bends

-Turn
(connect successive strands of antiparallel -sheets)

❑ Usual to find proline and glycine in -turns.

Coil or Loop
Frequency of amino acids in secondary structures
Learning: number of specific amino acids in different secondary structures
is different
Triple Helix
Learning: this is not the same as alpha helices

❑ Limited to tropocollagen molecule ( 3 strands of

molecules subunit)

❑ Sequence motif of –(Gly-X-Pro/Hypro)n-

❑ 3 left-handed helices wound together to give a
right-handed superhelix.
❑ Stable superhelix : glycines located on the
central axis (small R group) of triple helix.
❑ One interchain H-bond for each triplet of amino N
acid – between NH of Gly and CO of X in C C
C
adjacent chain.
O
Principles of structure-function
Learning: biological functions are related to 3D structures

❑ Function depends on structure

❑ Structure depends both on amino acid sequence & on weak, non-covalent forces
❑ Number of protein folding pattern is very large but finite

Unstable, unfavorable structures Most stable structure - favorable

 Slight changes can affect structure-function

Structure of the yeast guanylate kinase enzyme (Gkenz:) serine to proline mutant.

Mutation

Spindle-orienting function Catalyzes phosphotransfer from ATP to GMP

PNAS November 1, 2011 vol. 108 no. 44 E973-E978

 DOMAINS (modules)
Learning: many large proteins contain several discreet, independently folded
globular units – domains

❑ Each domain typically consists of about 100-200 aa residues and a combination

of motifs (Signatures. Describe the type of structures e.g. β motifs, β motifs)
❑ Have a specific function
B domain

A domain
Pyruvate kinase, a protein
with 3 domains

C domain
 Pyruvate Kinase

❑ fructose 1,6-bisphosphate (FBP) (red).

❑ The active site is located between the effector domains A and B where the
substrates phosphoenolpyruvate (PEP) and ADP (not in the structure) bind
together with K+ and Mg2+ (shown in pink).

❑ The C-terminal domain contains the FBP binding site

Green portion of protein can exist by itself

Trends Biochem Sci Volume 37, 309–316, 2012

Glyceraldehyde-3-phosphate dehydrogenase
❑ Exist as tetramer with a total MW 145 kD.
❑ Has 2 distinct domains, and NAD+ binds to the first domain while G-3-P, the
second.

Domain (/ motif) binds glyceraldehyde-3-phosphate

(not shown)

Domain binds NAD+

❑ Many domains are independent structural units and often have distinct functions
Certain domains are repeated in many proteins

Complement control Immunoglobulin Fibronectin type I Growth factor Kringle domain

Multiple domains make up some proteins

tPA 3D structure

Immunoglobulin
domain

Complement domain
Kringle domain

Growth factor domain

Fibronectin domain
Antibody structure
❑ Immunoglobulin domains contain about 70-110 amino acid.

Fab regions

Fc regions

Text

❑ Characteristic immunoglobulin fold: 2 beta sheets create a “sandwich”

shape, held together by interactions between conserved cysteines and other
charged amino acids.
 Structural Organization of Proteins
Learning: shape can be described at 4 levels of organization
Protein motifs are generally seen in ‘super secondary’ structures (between primary and secondary)
Domains are generally seen in tertiary structures
Different
proteins
Primary Secondary Tertiary Quartenary
linear arrangements local folds global folding aggregation of
global folds

Modified Nelson & Cox

 Quarternary structure
Learning: assembly of independent polypeptides stabilizes by
non-covalent interactions

❑ Multimeric proteins can have 2 – 100’s of subunits

❑ For example, mammalian haemoglobin has 2 (141 aas) and Tetrameric structure of
hemoglobin
2 (146 aas) polypeptide chains but earthworm Hbs have >
100 subunits

Some examples of proteins

with a single type of subunit

Structure-rasmol
Quaternary structure: advantages

❑ Oligomers more stable than dissociated subunits – prolong life of

protein in vivo.

❑ Active sites formed by residues from adjacent chains

❑ Error of synthesis is greater for longer/bigger chains. Therefore make

smaller once and the assemble them together.

❑ Subunit interactions : cooperativity/ allosteric effects – function of single

subunit is influenced (positively or negatively) by other associating
subunits.
 Protein families
Learning: some proteins share similar primary sequences and/or have similar
structures/ functions – same family.
Classified based on:
- Primary sequences
- Similar functions (despite ~50% of primary sequence being different)

❑ C-type cytochrome of different species – same protein family (homologues)

❑ same functions (electron carrier)
❑ low primary sequence similarity but structurally similar (specially in the interior of
the protein)
An example of an abundant protein -
Collagen
 Collagen: Structure-Function

❑ Most abundant protein (25-35% of total protein in mammals)

o Found in bones, tendons, skin, blood vessels, and cornea of the eye (different
structures and textures)
o ~30 genes encode collagen. >19 distinct types of collagen in human.
o Sequence motif of chain: Gly-X-Pro/Hypro

❑ Undergoes extensive post-translational modifications

o Hydroxylation of Pro and Lys residues (requires vitamin C/hydroxylase enzyme)
o Hydroxyproline – provide additional H-bond for stability of helix structure
o Hydroxylysine – attachment sites for carbohydrate moieties (glycosylation)

Collagen – a commonly used/abused in Cosmetics.

Organization of collagen

Collagen
fibrils

3 helices (triple helix)

Arrangements of Each helix
wound together
triple helices
(not  helix)
Modification of proline in collagen
Learning: hydroxyproline formed by post-translational modification requiring co-
factors (Vit C)

(Vitamin C)

Need to know: Proline can be

hydroxylated to form hydroxyproline
- Vitamin C required
 Other modifications in collagen

❑ Essential for formation of strong collagen fibrils

❑ Oxidation of side chains of (NH2)Lys to form

(OH)allysine (an aldehyde)

o (OH)Allysine-lysine x-links : intermolecular

covalent x-links between tropocollagen
molecules
o Allysine-allysine x-links : intramolecular
covalent x-links within a tropocollagen unit
(unlike keratin in hair where disulphide forms
(cys) x-links, resulting in strong fibrils).

❑ Collagen diseases : mutation of glycine is lethal

(gly-X-pro)

Covalently X- linked
Diseases associated with collagen
❑ Scuvy (not a genetic disease) – affects collagen x-linking. Deficiency of ascorbic
acid. Bleeding in gums, poor wound healing.
❑ Osteogenesis imperfecta results in abnormal bone formation in babies
❑ Ehlers-Danlos syndrome is characterized by hyperextensibility of skin, abnormal
tissue fragility, and increased joint mobility.

ED syndrome.MTS

❑ Both can be lethal: due to substitution of a Cys and Ser residue for a Gly in
collagen.

o Substitution (especially in C-terminus) produces catastropic effect by

disrupting the Gly-X-Pro (repeat) helical structure of collagen
Unscrambling & folding protein structures
Protein denaturation
Learning: irreversible disruption of native structure causes loss of function

❑ Loss of biological activity

❑ Conformational/structural change

❑ Heat, pH, organic solvents, urea, guanidine hydrochloride

❑ Destruction of 2o, 3o and 4o structures, but 1o structure remain intact

❑ Ease of proteolytic digestion, aggregation

❑ Reversible or irreversible
Denaturation of ribonuclease A
Learning: correct refolding conditions is necessary to regain functions

❑ Enzyme (124 aa) – digest RNA.

❑ Denatured by: 6 – 8M urea + 2 mercaptoethanol (reducing
agent)
❑ No activity
❑ Two routes for dialysis: either to remove urea or 2-
mercaptoethanol first
❑ Removal of urea first led to recovery of activity
❑ Information for folding resides in primary structure of
the protein

To unravel protein (re)folding is a major area of research

(Ribonuclease A study won Anfinsen a Nobel prize 1972).
 Denaturation-Renaturation of RNAse A
Learning: in vitro refolding is dependent on innate ability to fold correctly

Native state:
catalytically active

❑ 8 Cys.
❑ Probability of any 4 Cys forming the
native form of disulphide randomly-
1:105. Unfolded state:
Inactive. Disulfide
❑ Renaturation results in fully active cross-links reduced
protein – refolding results in mostly to yield Cys
residues.
correct native form of the protein.
Conclusion: primary sequence dictate 3-
D structure for this protein.
Native state:
catalytically active
state. Disulfide links
correctly re-formed.
 Protein folding process
Learning: protein folding is not a random process, or it will take too long to
correctly fold

Unfolded Folded

❑ A 100 residue protein : random search of all possible conformations will take
4x109 years – Levinthal’s paradox

❑ A cooperative and sequential process : formation of one part of the structure

leads to the formation of the remaining structure

❑ Folding pattern and final conformation depend on primary structure

DrKjaergaard
Stages in protein folding
2° structures formation
Some 2° structures like -helix form
early and are quite stable

Unfolded
No structure

Molten globule stage

Different parts of the protein
have come together in a correct
configuration forming a “glob”

Folded
Stable form of protein
Observation: process of denaturation, can identify
“molten globule” (intermediate state)
Recombinant insulin - biotechnology
Learning: proteins can be manufactured using heterologous cells

Cellular processing of insulin

❑ Originally isolated from pig pancreas (1920s)- 1 pancreas/patient/3 days

❑ slightly immunogenic – human raise antibody to pig insulin
❑ Human insulin: 1970s advent of molecular biology – cloned human insulin
Biotechnology and human insulin
Insulin manufacturing

❑ Produced in genetically engineered bacteria

(E.Coli) to express A/B chains (as a form of
fusion protein with gal)
❑ Subsequently denatured/renatured to form the
correct disulphides and folded protein.

Figure after © 2000 by Griffiths et al.

Conditions for refolding in vitro
Learning: refolding in vitro require space so as not to allow denatured forms
to aggregate

❑ Buffer + denaturant + reducing agent (e.g., dithiotheritol)

❑ Low temperature
❑ Large volume (low concentration of denatured protein; < 0.1 mg/ml)
❑ Slow Dialysis to remove denaturant and allow gradual refolding

No SPACE in cells as compared to in vitro

Providing an environment that allows individual molecule to

refold.
No crowding!
How crowded is the cell?

200-300 mg/ml proteins

❑ How does a protein find

another to interact?

❑ If denatured will these

aggregate and be insoluble?
Proteins are folded correctly inside cells
Learning: cells can fold in crowded environments & fast

Nucle
us
Nucleus

mRNA exporting into the

cytoplasm Rough
Endoplasmic
reticulum (RER)
Some proteins are
translated and stay in
the cytoplasm

Protein folding is
assisted by chaperones

Inside of the RER

 Molecular chaperones: folding machinery
Unfolded proteins usually possess numerous solvent-exposed hydrophobic regions
o Tendency to self-aggregate especially in concentrated solution (e.g, inside a cell)

❑ Function to prevent or reverse such improper associations by binding to these

hydrophobic areas
❑ Subsequent release of these proteins will facilitate proper folding based on their
amino acid sequence.

❑ These include peptidyl prolyl cis-trans-isomerases

(PPIs) & protein disulphide isomerases (PDIs) –
improves folding of proteins
o PPIs: catalyze the isomerization of incorrect Xaa-Pro bonds
which is a slow rate determining step Molecular chaperone
o PDIs: facilitates the formation of correct set of S-S bonds

❑ Chaperones – act as
o “Molecular incubators” (Not catalysts)
o Enzyme to ‘redo’ (e.g., PDI)

Model of hsp60 family of molecular chaperone

 Protein folding & Denaturation in vivo (inside a cell)
Learning: cellular mechanisms for assisting protein folding/refolding

❑ Molecular chaperones (specialized

proteins) help fold proteins inside cells.

❑ Misfolding can result in death of cells – E.Coli deal by forming inclusion bodies.
Mammalian cells cannot form insoluble products inside. They then commit
suicide (apoptosis)
 Intracellular quality control of protein folding
Learning: cell has mechanisms to deal with unfolded proteins
Unfolded protein
response (UPR) –
transcriptional Ubiquitin (76 aa protein) + ATP
activation of stress
proteins (chaperones)

EMBO reports (2005) 6, 1131 - 1136

Misfolding can result in cell death
Learning: when quality controls fail, aggregation can
result in stress which leads to death

Prion (proteinaceous infectious agent) Diseases [Transmissible

spongiform encephalopathies (TSEs)]

❑ Kuru was once found among the Fore tribe in Papua New
Guinea. (“laughing deaths” – eating the dead ritual)

❑ ‘Mad cow disease’ (Bovine Spongiform Encephalopathy)

outbreaks – cow feeds. Transmissible to human eating
contaminated meat

❑ Fatal.
 Single copy gene in chromosome 20 – natural form PrPc.
❑ Prion (protein), PrP, has a molecular mass of 28,000 dalton.
❑ Found in the brain tissue of all mammals, but function unknown.
Seen in the synapse— likely to do with neurotransmission
Normal Diseased

Altered form –
insoluble fibrils
3  helices + 2 short
antiparallel  sheets

Healthy prion protein folds Diseased prion folds a portion of itself

itself into a series of helices into strands and sheets

Protease sensitive Protease insensitive

Cayman chemicals
Examples of diseases associated with protein misfolding

Therapeutics involved:

❑ Prevention of formation of misfolded proteins

❑ Removal of misfolded proteins
CurrPharmDesign. Vol12, 2006
Summary of Topics I/II

Journey to understand

❑ How chemical forces, pH & water interact with biomolecules

❑ How proteins are built, attain structures and function