Topic II: AMINO ACIDS, PEPTIDES & PROTEINS

insulin

 Amino acids – structures & properties.

 Peptides.
 Proteins –
o primary, secondary, tertiary & quaternary structures.
o protein folding
 What do proteins do in a cell?

Proteins are “workhorse” of cell processes

Alberts et al

Examples:
1. Structural functions – proteins acting 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 ~ $30-60 (USD)/g

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

 Best selling Drugs are biopharmaceuticals

Produced using cells

Biopharmaceuticals

Pharmaceutical
 Parkinson’s disease and celebrities……….

“Dance like a
butterfly and
stings like a bee”…

Michael J Fox foundation

Parkinson’s disease – the “ghost” within.
L-dopa
A promising protein to “cure” Parkinson’s disease?

GDNF dimer

 GDNF : used to produce dopaminergic neuron from embryonic

& inducible stem cells for cell therapy (experimental)
Clinical benefit of GDNF infused into the brain.
Biomolecules have complex molecular structures

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

Tissue plasminogen
Insulin hexamer activator
(Diabetes M) (acute myocardiac
infarction)
 Why study the structures of proteins?

 Structure of proteins correlates with functions of the proteins

 Changing structure of the protein may 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
 Critical ASsessment of Techniques for Protein
Structure Prediction (http://predictioncenter.org/)

 13th Community Wide Experiment (14 Dec 2018)

 Started in 1994

 May - July 2018, CASP organizers posted sequences of unknown protein

structures for modeling.

 Modelling is getting more accurate

 Each “key and lock” (protein-protein
interaction) sets have different 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 do different proteins adopt
different conformations?
 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

 Same compositions of amino acids but different arrangements – may

give rise to different conformations of the proteins
 Amino acids are building blocks of Proteins
R O R O
+ -
H2 N CH C OH H3 N CH C O Amino acid

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

Zwitterion

 Proteins are polymers of amino acids

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

Polypeptide chain

Peptide bond
 AMINO ACIDS – Building blocks of proteins

 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)

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

R
 Thus, amino acids have stereoisomers.

R In cells: predominantly L-amino acids exist.

H2N COOH
H R

H2 N H

COOH
H COOH

NH2

Fingers/tumb to illustrate asymmetry.

 Amino acids condense to form peptides & proteins

 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
 Depending on the physicochemical properties of the R-group, they can be
classified into :
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

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

Neurotransmitter

Not a good nucleophile (NH2)

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

 Important “excitatory” neurotransmitters

(epilepsy) – chinese restaurant syndrome
 POLAR basic side group

Animal feed

+ +
 Amino acids can also be grouped –
‘essential’ & ‘non-essential’

 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

Plants/microbes make all these. Histidine Asparagine

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 are found at different
frequencies in proteins
 3D Structures of amino acids

Glycine Alanine Phenylalanine

 Proline – cyclic amino acid. Conformational constraints

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

Illustration – rasmol.
 Acid-Base properties 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 = pKa/2 ; pKa of groups that will pI = 9.6 + 2.3

lose/gain charge of the neutral species. 2
= 5.9
 Titration of Glutamic acid

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

pK2 9.2

pI = 9.2 + 10.8

pK1 2.2 2
=10
 Other naturally occurring amino acids
found in proteins
 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).
 Another rare naturally occurring
amino acids found in proteins

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

amino group.

 
 Lysine
 

Pyrrolysine

 Special tRNA used for biosynthesis.

 Encoded by a UAG codon (normally a stop codon).
 Modified amino acids found in some proteins
 Some amino acids in certain proteins can be 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.
 Hydroxyproline

Hydroxylated form of proline

 Found in collagen

 Provides stability to collagen fibers

 Insufficient hydroxylation leads to scurvy- due to lack of vitamin C

 Modified amino acid





Protein
Glutamic acid -Carboxyglutamic acid

 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

 Modified amino acid

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.
Post-translational modifications of amino acids are
involved in cell signalling

 Cells convey signals from external to internal by exploiting

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

PO4- added to tyrosine on receptors

Cell signalling involves relay of information

 Involves relaying information

from protein to protein

* For information only

Other post-translational modifications

Acetylation (histones)

Myristoylation

Palmitoylation

Prenylation Adenylylation
ADP-ribosylated histidine
There are some derivatives of amino acids that are
Not found in proteins

Glutamic acid Histidine Tryptophan

Metabolism
 Amino acids, 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”
 Biologically active peptides
 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.
 Naturally occurring peptides with
anti-microbial activities

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)
 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 predicted from the genome project

Functions are still unknown.

(only know about 50%)
Enzymes
 Proteins: large and small
 Too complex to 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)

 Proteins group 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 casting
to sample loading
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

 Intracellular proteins
 Extracellular proteins PERIPHERAL PROTEINS:
Proteins that are associated
with the membrane

INTEGRAL PROTEINS

LIPID ANCHORED PROTEINS:

Proteins linked via lipids to the
membrane

Receptors, transporters
Modified Karp
 Globular shaped Proteins
 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
 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
 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
 Structural Organization of Proteins
4 levels of organization

Primary, Secondary, Tertiary & Quaternary Structures

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

Secondary structures

-Turns

-Sheets

Loops

-Helix
Various non-covalent forces stabilize protein structure

 H-bonds

Hydrophobic residues buried inside molecule  Hydrophobic interactions

(shielded from the environment)
 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

 Peptide bond in its usual trans conformation of carbonyl O and amide H

Peptide bond characteristics

 Delocalized bonds result in shorter bond

length and result in a rigid bond – amide
bond has partial double-bond character.
 Amide bond properties

 Dipole generated by resonance structure-slightly polar

 Non-chemically reactive – N cannot react as a nucleophile (electron donor)

 Due to rigid configuration, C-C-N-C rotates

as a plane – coplanar relationship of
atoms in amide group
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

 Most combinations of  &  angles are sterically forbidden.

 Structures are constrained by the amide planes

N- terminus
C- terminus

Bond is not free

to rotate

 Calculate the  &  angles for every residue –

Ramachandran plot (conformation plot)

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 and -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
 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

Parallel H
N

Antiparallel
 Helix- sheet composites in spider silk
 Stronger than steel and tougher than Kevlar yet flexible

Composites of α-helices and β-sheets

 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
 Triple Helix

 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
acid – between NH of Gly and CO of X in
N
adjacent chain. C
C
C
O
 Biological functions are related to
3D structures of proteins

Principles of structure-function
 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)

 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 (PDB/1PKN)

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

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 structures/domains are
repeated found in many proteins

Complement control Immunoglobulin Fibronectin type I Growth factor Kringle domain

 Domains/modules 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

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

shape, held together by interactions between conserved cysteines and other
charged amino acids.
 Quarternary structure

 Stabilize by non-covalent interactions between subunits

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

 For example, mammalian haemoglobin has 2 (141 aas) and

2 (146 aas) polypeptide chains but earthworm Hbs have > Tetrameric structure of
hemoglobin
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
 Proteins sharing similar primary sequences and/or have similar structures/
functions – same family.

 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)
 Structure-Function: Collagen (example)

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

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

 Undergoes extensive post-translational modifications

 Hydroxylation of Pro and Lys residues (requires vitamin C/hydroxylase enzyme)
 Hydroxyproline – additional H-bond for stability of helix structure
 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

(Vitamin C)
 Other modifications in collagen

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

(OH)allysine (an aldehyde)

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

covalent x-links between tropocollagen
molecules

 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).

 Essential for formation of strong collagen

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.

..\Movies\ED syndrome.MTS

 Both can be lethal: due to substitution of a Cys and Ser residue respectively for a
Gly (different Gly for each) in collagen.

 This substitution (especially in C-terminus) produces catastropic effect since it

disrupts the Gly-X-Pro (repeat) helical structure of collagen
 Protein denaturation

 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

 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

Native state:
catalytically active

 8 Cys.
 Probability of any 4 Cys forming the
native form of disulphide randomly- Unfolded state:
Inactive. Disulfide
1:105. cross-links reduced
 Renaturation results in fully active to yield Cys
residues.
protein – refolding results in mostly
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

Unfolded Folded

 Not a random process

 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
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

 Produced in bacteria (E.Coli)

 Initially produced by genetic engineering of A and B chains separately and
then denature/renature to form the correct disulphides.
 Folding of a mature polypeptide (e.g., insulin with A-B chain) can be assisted
by the presence of polypeptides (e.g., chain of insulin).

Genetic engineering to produce proinsulin

Denaturation/refolding/oxidize
Enzymatic
cleavages
proinsulin Insulin – folded correctly
In vitro.
 Conditions for refolding in vitro

 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

Providing an environment that allows individual molecule to

refold.
No crowding!
 Proteins are folded correctly inside cells
Nucleus

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

 How crowded is the cell?

200-300 mg/ml proteins

How does a protein find

another to interact?
 Molecular chaperones: folding machinery
 Unfolded proteins usually possess numerous solvent-exposed hydrophobic regions
 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) and protein disulphide
isomerases (PDIs)
 PPIs: catalyze the isomerization of incorrect Molecular chaperone
Xaa-Pro bonds which is a slow rate
determining step
 PDIs: facilitates the formation of correct set of
S-S bonds
 Chaprones

Model of hsp60 family of molecular chaperone

 Protein folding & Denaturation in vivo (inside a cell)

 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

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
e.g., Prion, Alzheimer’s disease & other
neurodegenerative diseases.

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).

 Misfolded protein ‘cause’ several rare degenerative

brain diseases.

 ‘Mad cow disease’ outbreaks – cow feeds.

 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.

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