Outline of the Lecture
Objective
Emergence of Life
Lecture 1&2
Acknowledgements:
Alberts - Molecular Biology of the Cell
Scitable by Nature Education
Nature Resources
Internet resources
Was life supposed to evolve?
nWhat we normally think of as life is based
on chains of carbon atoms with a few
other primary atoms such as oxygen,
hydrogen, nitrogen and phosphorous.
nWhat about silicon-based life?
n Yes, maybe! but carbon-based seems
the most favorable because it has the
richest chemistry
The Anthropic Principle:
For a given universe, it is possible that the
values of the physical constants, will allow
the existence of objects like carbon atoms
that can act as the building blocks of
living systems
vRetrace the assembly of matter to form life
Topics
vIntroduction and background
vSome scientific/philosophic questions
vConcept of order
vPhysical, Chemical and Biological Parameters
v Information encoding in biomolecules
vThe “creation” of the cell
1 2
Why did life occur?
vWe don’t have an answer! Its like asking – Does
God Exist?
vThe anthropic principle tells us that LIFE would
have happened if not on planet earth, then on
some other planet
vWe do know, by observation, what are the
properties that define life
vHave a set of instructions that tell the system how to
sustain and reproduce itself
vA mechanism to carry out the instructions
vBy the second law of thermodynamics, to do
this, we need to have order
https://www.youtube.com/watch?v=KtRcAuunEMg
3 4
 What is the role of “Order” in the The history of life
creation of the first cell?
vAnd it took 2.5 billion years for life to
nHow do you determine if a system has order - in the evolve from the earliest cells to multi cell
sense of structured-ness and organization ? animals
oSelf Organization vAnother 1 billion years to evolve
oComplexity through fish and reptiles to mammals
oEmergence of new properties vThen evolution seemed to have speeded
n One of the most fundamental problems in biology concerns the origin up
of forms and their associated functions
vtook about a hundred million years to develop from
n It has been an important question of developmental biology
the early mammals to us
Third law of Thermodynamics states that the entropy (disorder) of
the universe is continuously increasing with time.
Therefore, decrease of entropy, as in a cell, is permitted if the
corresponding increase in entropy in the environment is greater.

5 6

We will examine how “Order” occurs Order in inanimate systems: Physical Systems

In-animate Systems
vPhysical systems
vChemical systems
vBiological self-assembled systems
a) In a liquid layer, molecules are agitated by thermal motion.
b) The molecules in the liquid layer are heated from below (red zone) and
Living Systems self-organize into rolls (drawn in cross-section) when the temperature
reaches a critical value (tc). At this value, the molecules start to move
vThe cell collectively either up or down at point 0, which determines the
alternative orientation of the rotation of the rolls throughout the layer.
The orientation of the rotation choice is unpredictable and determined
by local fluctuations at tc
7 8
 Self-assembled systems that occur in Biological world
The boundary: inanimate to animate
Order (Self Organization) in Chemical Reactions
In the system, propagation of a vThe concepts of interaction free
single wave was observed
under following conditions: energies, molecular geometry and
entropy, when taken together,
NaBrO3 = 0.23 M, bromomalonic furnish a framework for a theory of
acid = 0.16 M, (Ferroin) self-assembly or self-organization
Fe(phen)3 = 0.003 M, H2S04 =
0.26 M, T = 14°C. vMicelles and bilayer vesicles
The wave velocity equalled assembly are driven by hydrophobic
approximately 0.01 cm/s. and hydrophilic interaction - of two
'opposing forces"
vSize of the micelle is determined by
Photographs taken at 30 s In class demonstration of the B-Z reaction "optimal surface area" per head
intervals https://www.youtube.com/watch?v=o72GGxQqWt8 group at which the total interaction
free energy per lipid molecule is a
Belousov–Zhabotinsky reaction J. Theor. Biol.
(1973) 40, 45-61
minimum.

9 Biochimica et Biophysica Acta, 470 (1977) 185--201 10

Some basic rules for micelle shapes

Vesicle Size and Shape
Adv Mater 2003 15 (16)

Adv Mater 2003 15 (16)

• Surfactant first forms a bilayer and then closes to

form a vesicle
• The ratio of the hydrophobic to hydrophilic portion
of the molecule determines the radius of curvature
at the interface
vDepends on mixing entropies (pull towards AFM image of a polymer
v Kl 2 many assemblies) and molar bending vesicle. The erythrocyte-
= 1 + Hl + like shape arises from the
al 3 energies ( tends towards a smaller number evaporation of water from
of vesicles) the vesicle interior,
vDA is the difference in area, v is the leaving the prominent rim

11 dimensionless volume to area ratio

12
Similarity in structure of self-organized biological and non-biological
entities
(a) High-genus block copolymer
vesicle (b) structure of a diatom
[Adv Mater 2003 15 (16)]
vGenus order g > 100
Urey Miller Experiment
vIn 1953 Stanley Miller and Harold Urey
performed the first experiment that
produced amino acids in what was
assumed to be a pre-life atmosphere.
They passed a mixture of water vapor,
DMPC–apoE CT domain methane, hydrogen and ammonia gases
complexes; reconstituted
lipoprotein particles were
through an electric arc to simulate what
TEM image showing
ordered chains of
stained with 2% would happen if these gases were
phosphotungstate for
prismatic BaCrO4
visualization [Biochem. J.
subjected to lightning.
nanoparticles prepared in
(2005) 387 (747–754) ]
AOT microemulsions Result:
Angew. Chem. Int. Ed.
2003, 42, 2350 – 2365 v10 biologic amino acid types
25 non-biologic amino acid types
Formaldehyde
Sugars

13 14

First proteins Formation of polynucleotides and polypeptides

vIn the presence of kaolinite, amino acids are
picked up from an aqueous solvent and
brought into solid solution
vAmino groups become hydrogen
bonded to structural oxygen
vIn water, amino acids cannot polymerize
because of dipole-dipole interactions
vIn solid solution, however, amino acids
will polymerize, because the solvent
medium does not interfere
vabout 1000 times more amino acids
were polymerized to peptides
vKaolinite is also instrumental in
preferentially synthesizing pentoses and
hexoses from formaldehyde and v Nucleotides of four kinds (here represented by the single letters A, U, G, and C)
transforming them into polysaccharides can undergo spontaneous polymerization with the loss of water. The product is
vPreferential polymerization of L-amino acids a mixture of polynucleotides that are random in length and sequence.
on kaolinite can be attributed to the v Similarly, amino acids of different types, symbolized here by three-letter
inherent enantiomorphism of the edges of abbreviated names, can polymerize with one another to form polypeptides.
the octahedral layer of kaolinite Present-day proteins are built from a standard set of 20 types of amino acids.

15 16
 Replication of a polynucleotide sequence
Polynucleotides as templates

vThe original RNA molecule acts as a template to form an RNA molecule

of complementary sequence.
vThis complementary RNA molecule itself acts as a template, forming
RNA molecules of the original sequence. Since each templating
vPreferential binding occurs between pairs of nucleotides (G molecule can produce many copies of the complementary strand,
with C and U with A) by relatively weak chemical bonds these reactions can result in the "multiplication" of the original
sequence

17 18

Conformation of an RNA molecule Evolutionary significance of cell-like compartments

vAny improved form of RNA that is able to promote formation of a more

useful protein must share this protein with its neighboring
competitors.
vIf the RNA is enclosed within a compartment, such as a lipid
membrane, then any protein the RNA causes to be made is retained for
its own use; the RNA can therefore be selected on the basis of its
guiding production of a better protein.
19 20
Central Dogma Last Universal Common Ancestor
Suggested stages of vThe evolution of the
evolution from simple translation apparatus
occurred in a series of
self-replicating systems increasingly complex stages,
of RNA molecules to rather than all at once,
present-day cells. Today,
DNA is the repository of vThe stages subsequent to the
establishment of the basic
genetic information and mechanism were concerned
RNA acts largely as a go- by and large with increasing
between to direct the mechanism's accuracy,
protein synthesis and possibly speed as well
J. Mol Evol 10, 1-6, 1977

21 22

Evolution of Metabolic Pathways Predation to new cellular structures

v The cell on the left is provided with a
supply of related substances (A, B, C,
and D) produced by prebiotic synthesis. vA close relative of
One of these, substance D, is
metabolically useful. As the cell present-day
exhausts the available supply of D, a cyanobacteria that lives
selective advantage is obtained by the
evolution of a new enzyme that is able in a permanent symbiotic
to produce D from the closely related relationship inside
substance C. Fundamentally important
metabolic pathways may have evolved another cell. The two
by a series of similar steps. organisms are known
v On the right, a metabolically useful jointly as Cyanophora
compound A is available in abundance.
An enzyme appears in the course of paradoxa
evolution that, by chance, has the ability
to convert substance A to substance B. vThe "cyano-bacterium" is
Other changes then occur within the cell in the process of dividing
that enable it to make use of the new
substance. The appearance of further
enzymes can build up a long chain of
reactions.

23 24
Animal and Plant Cell structures Cellular Membrane System

25 26

Summary Inner Life of a Cell

vAutocatalytic mechanisms fundamental to living systems began with the

evolution of families of RNA molecules that could catalyze their own replication.
vFamilies of cooperating RNA catalysts developed the ability to direct synthesis of
polypeptides.
vAccumulation of additional protein catalysts allowed more efficient and complex
cells to evolve, the DNA double helix replaced RNA as a more stable molecule for
storing the increased amounts of genetic information required by such cells
vPresent-day living cells are classified as procaryotic (bacteria and their close
relatives) or eucaryotic.

Full version
27 https://www.youtube.com/watch?v=FzcTgrxMzZk
 “Life is an emergent, rather than an
inherent, property of matter. Although it
arises from the material world, it cannot be
Fundamental Units of Life reduced to it”

All organisms are like a machine; while a

Lecture 3
machine implies a machine maker, an
organism is a self-organizing entity

SURELY YOU MUST BE

JOKING MR FEYNMAN

1 2

Sizes of Things
Outline of the lecture

Objective: to learn about Atom

the main biomolecules
that are part of the
building blocks of cells
vCarbohydrates and
Lipids – cell structure
vAmino acids – proteins
vNucleotides – DNA and
RNA

Look at some applications

3 4
 Chemical composition of the cell, the main Fatty Acids
biogenic elements.
Saturated fatty acids are relatively simple lipids with the general
formula
CH3¾(CH2)n¾COOH
The value of n is typically between 12 and 20 (Even Numbers in
biological systems)
Stearic Acid (saturated) : CH3 ¾(CH2)16¾COOH
Oleic acid (unsaturated):CH3 ¾(CH2)7 ¾HC ═ CH¾(CH2)7 ¾COOH
vThe bulk of cell’s mass is v Carbon containing compounds
made up by water are degraded to CO2 and H2O
by combustion, mineral
compounds remain
5 6

Configurations of Fatty Acids in Water

Fats
Polar head nonpolar hydrocarbon tail Fats are condensation products of fatty acids and
glycerols (esters)
AIR O
CH2OH HO¾OC(CH2)n¾CH3 CH2O¾C¾(CH2)n¾CH3
+ O
CHOH + HO¾OC(CH2)n¾CH3 - 3H20 CHO¾C¾(CH2)n¾CH3
+ O
CH2OH HO¾OC(CH2)n¾CH3 CH2O¾C¾(CH2)n¾CH3

Lipid monolayer at the air water interface Lipid micelle in water glycerol Fatty acids Fat

Lipid molecules have a very small solubility and elevation of the solution
concentration above the monomolecular solubility results in the condensation
of the excess solute into larger ordered structures called micelles (DG < 0)
7 8
 Monosaccharides
Monosaccharides
vD-glucose is the most common
ALDOSES KETOSES monosaccharide found in living
CHO CH2OH organisms 5
CH2OH O
TRIOSE
HCOH C═O
vSimple sugars are found either in
CH2OH CH2OH the aldehyde or keto form: For 4 1
D-glyceraldehyde Dihydroxyacetone
example, glucose is an aldohexose OH
CHO CH2OH
D-ribose
HCOH C═O vD- is the optical isomer almost 3 2
PENTOSE OH OH
HCOH HCOH exclusively found in living organisms
HCOH HCOH 5
6 CH2OH O
CH2OH CH2OH CH2OH
D-ribose D-ribulose 5 4 1
CHO CHO CHO CHO O b
HEXOSE
HCOH HOCH HCOH C═O OH
HOCH HOCH HOCH HOCH
4 OH 1 deoxyribose 3 2

HCOH HCOH HOCH HCOH HO a OH

HCOH HCOH HCOH HCOH 3 2
p The five membered rings, D-ribose and deoxyribose are
CH2OH CH2OH CH2OH CH2OH 9 OH the primary components of the nucleic acid monomers
D-glucose D-mannose D-galactose D-frucose D-glucose DNA and RNA 10

Polysaccharides – Starch and Cellulose Polysaccharides

vAmylose – continuous a-1,4-glucosidic bonds

vStorage material vDraw the structure of amylose
vAmylose is straight water insoluble
vAverage molecular weight of amylose is 0.5–1 ´ 106 vDraw the structure of amylopectin
vAmylopectin is crosslinked polymer vDraw the structure of cellulose
vwith branches occuring every 25 glucose units by condensation with
the C6 –OH group C
O
C
O
C
O
C
O
C
O
C
O

vAverage molecular weight of amylopectin is 1–2 ´ 106 O O O O O

O

vGlycogen – storage material in animals (livers) C

O
C
O
C
O

vGranules having amylopectin structure but more cross-linked; almost O O O

every 12 glucose units C

O
C
O
C
O

vCellulose – continuous b-1,6-glucosidic bonds O O O

vStructural material

11 12
Brown Rot Versus White Rot fungi Biorefinery using wood as feed-stock

Cellulose is eaten away Lignin is eaten away

Phanerochaete
chrysosporium
https://doi.org/10.1016/B978-0-12-813056-8.00005-4
13 14

Nucleotides, RNA and DNA

Production of Bioethanol
Nucleotides
vPresent in nucleic acids
vMade up of three components
vPhosphoric acid
vRibose or deoxyribose 5-C sugars
vNitrogenous base either purine or pyrimidine
Base
OH Base
OH
OH P O CH2 O OH P O CH2 O
O H H
O H H
H H H H
OH H OH OH

Deoxyribonucleotide ribonucleotide
15 16
 Purines and Pyrimidines Nomenclature of Nucleosides and Nucleotides
DNA only DNA & RNA RNA only
H
N
H
O Base Nucleoside Nucleotide
N N H
N N
Purines

H H Adenine (A) Adenosine Adenylate (AMP)

H
N H N N
N N
H H H
Cytosine (C) Cytosine Cytidylate (CMP)
Adenine Guanine

H H
Guanine (G) Guanosine Guanulate (GMP)
O N O
Pyrimidines

H3C H H H H
H
N N N
Uracil (U) Uridine Uridylate (UMP)
H N O H N O H N O

H H H Thymine (T) Deoxythymidine Deoxythymidylate dTMP)

Thymine Cytosine Uracil
17 18

The Phosphates of Adenosine ATP the Energy Currency

Adenine Adenine
OH OH OH
OH P O CH2 O OH P O P O CH2 O vThe conversion of ATP to ADP
H H H H
O
H H
O O
H H and phosphate is accompanied
OH OH OH OH by a standard free energy of –7.3
Adenine Monophosphate (AMP) Adenine diphosphate (ADP)

Adenine
kcal/mol at 37oF and pH 7
OH OH OH
Adenine
O CH2 O vEnergy derived from nutrients or
OH P

O
O

O
P O P

O
O CH2
H
O
H H
H H
H
sunlight is stored as ATP in cells
H
OH OH
H O P O OH vCyclic AMP serves as a regulator
OH
Adenine triphosphate (ATP) Cyclic AMP in many cellular reactions
v AMP, ADP and ATP are important in cellular energy transfer processses
v Cyclic AMP serves in regulatory functions

19 20
 The ATP-DNA Paradox Co-enzymes Derived from Nucleotides
Adenine Adenine Adenine

ATP is synthesized by a O CH2 O O CH2 O O CH2 O

H H H H H H
molecular machine called H H H H H H

the ATP Synthase. This OH P

O
O OH OH OH
O
P O OH OH OH
O
P O OH OH

machine has to be built from OH P O OH P O OH P O

information contained in O O O

Riboflavin Nicotinamide Pantothenic

DNA. But converting this Acid
Flavin adenine dinucleotide (FAD) Nicotinamide adenine dinucleotide (NAD)
information and stitching Oxidized form b-mercapto-ethyleneamine

amino-acids together requires Co-enzyme A

Three important co-enzymes derived from nucleotides
ATP as energy currency.
DAO-FAD

21 22

Biological Information Storage Double Helix Structure of DNA

Base 1 Base 1
5’ end vJames Watson and Francis
CH2OH CH2OH
O O
Crick deduced in 1953, the
H H H H
H H H H
DNA molecule consists of
OH OH O two polynucleotide chains
P O
P O Base 2
coiled into a double helix
vThe regular backbone of
–
–O Base 2 O
OH CH2O O
CH2OH O H H the molecule is composed
H H H H of sugar and phosphate
H H O units
OH OH
P O

P O
–
O
Base 3
vIn the interior of the double
–O Base 3
CH2O O helix are the purine and
OH
CH2OH
H H pyrimidine bases
Nucleotides condense to form a chain O H H
linked by phosphodiester bonds H H OH
H H 3’ end
OH 23 24
 DNA Computing Chemical composition of the cell …

oAmino acids are the building blocks of proteins

oThere are 20 amino acids commonly found in proteins
oSimple proteins are polymers formed by the condensation of
amino acids by forming the peptide bond
25 26

Individual Properties of Amino Acids

L and D Forms of Amino Acids amino acid
mass
surface b volume c pKa d pI e solubility e density e

Alanine ALA A 71.09 115 88.6 - 6.107 16.65 1.401

Arginine ARG R 156.19 225 173.4 ~12 10.76 15 1.1

vThe L and D forms are not Aspartic Acid ASP D 115.09 150 111.1 4.5 2.98 0.778 1.66

Asparagine ASN N 114.11 160 114.1 - - 3.53 1.54

superimposable Cysteine CYS C 103.15 135 108.5 9.1-9.5 5.02 very high -

vGlycine where R=H is the only Glutamic Acid GLU E 129.12 190 138.4 4.6 3.08 0.864 1.460

exception Glutamine

Glycine
GLN

GLY
Q

G
128.14

57.05
180

75
143.8

60.1
-

-
-

6.064
2.5

24.99
-

1.607

vLiving organisms only have the L- Histidine HIS H 137.14 195 153.2 6.2 7.64 4.19 -

Isoleucine ILE I 113.16 175 166.7 - 6.038 4.117 -

form with very rare exception in Leucine LEU L 113.16 170 166.7 - 6.036 2.426 1.191

the cell wall of some bacteria Lysine LYS K 128.17 200 168.6 10.4 9.47 very high -

Methionine MET M 131.19 185 162.9 - 5.74 3.381 1.340

Phenylalanine PHE F 147.18 210 189.9 - 5.91 2.965 -

Proline PRO P 97.12 145 112.7 - 6.3 162.3 -

Serine SER S 87.08 115 89.0 - 5.68 5.023 1.537

Threonine THR T 101.11 140 116.1 - - very high -

Tryptophan TRP W 186.12 255 227.8 - 5.88 1.136 -

Tyrosine TYR Y 163.18 230 193.6 9.7 5.63 0.0453 1.456

Valine VAL V 99.14 155 140.0 - 6.002 8.85 1.230

a
mass [dalton], surface [Å2], volume [Å3], pKa [side chain], pI [at 25°C], solubility [g/100g, 25°C], density [crystal density, g/ml],
27 name: information from NIST Chemistry WebBook, three letter code: GIF, one letter code: VRML
28
 Amino Acid Table Proteins – Food Industry
The hamburger was made from 20,000
vThe R groups are muscle fibres grown from stem cells.
ionizable Non- Photgraph: David Parry/EPA
vHydrophobic, polar
Dr Post's team at Maastricht University. These
hydrophilic, acidic and fibres were extracted from individual culture
basic
wells and then painstakingly pressed together
vCondensation reaction to form the hamburger that was eaten. The
Polar
between the amino objective is to create meat that is biologically
group of one acid and
the carboxyl group of identical to beef but grown in a lab rather
another results in the than in a field as part of a cow.
formation of the
peptide bonds
Acidic Basic 29 30

Chemical composition of the cell … Elements of life…

Elements Symbols %
vThe concentration (%) of most
Oxygen O 65
Elements that make up life Carbon C 18
important elements (macro-
elements) in human body
v92 elements are found in nature (118 total elements) Hydrogen H 10
Nitrogen N 3
vLiving objects are composed of 25 – 26 elements.
Calcium Ca 1,5
Phosphorus P 1,0
Sulphur S 0,25
Potassium K 0,2
Sodium Na 0,15
Chlorine Cl 0,15
Magnesium Mg 0,05
Others 0,75

31 32
Elements of Life … Elements of life…
Approximate amounts of important microelements within 70 kg mass human body

Element Symbol Amount Role in the organism

vOther important microelements:
Iron Fe 4–5g Red-ox reactions; oxygen Lithium (Li) – regulation of nerve functions;
transportation within erythrocytes
Zinc Zn 1,4 – 2,3 g Regulation of the growth and Selenium (Se) – protein biosynthesis, hair;
development, synthesis of hormones
and proteins (hair, skin). Fluorine (F) – development of bones and teeth;
Copper Cu 75 – 150 Oxidation reactions, biosynthesis of
mg the skin pigment melanin Iodine (I) – hormone biosynthesis, neural regulation.
Manganese Mn 12 – 20 mg Formation of skin and mucous
layers, development of blood cells
Molybdenu Mo 5 – 9 mg Red-ox reactions at respiration Ultramicroelements:
m
Cobalt Co 1 – 1,5 mg Metabolic processes, the component Arsenic (As) and Gold (Au) – regulation of growth and metabolism
of vitamin B12
Chromium Cr 0,6 – 1,4 Sugar turnover, action of insulin
mg
33 34

35
Living organisms operate within laws of physics
and chemistry From Procaryotes
vConservation of Mass, Energy
vLaws of Chemical Kinetics to
vPrinciples of Chemical Reactions
Eucaryotes
vConverts energy to work
Lecture 4
vCatalyzes chemical transformations
vAssembles complex molecules from simple subunits.
vComplex molecules combine to form supra molecular
components, organelles and finally assemble into a cell
vCells store and pass on instructions for the assembly of all
future generations from simple non-living precursors

35 1
Procaryotic Cells Are Structurally Simple but
Biochemically Diverse The power of the lowly bacteria

v Bacteria are the simplest
organisms found in most natural
environments.
v Spherical or rod-shaped cells,
commonly several micrometers in
linear dimension Possess a tough
protective coat, cell wall, beneath
which a plasma membrane
encloses a single cytoplasmic
compartment containing DNA,
RNA, proteins, and small
molecules.
Procaryote sizes and structures. (A) Some procaryotic cells drawn to
scale. (B) Electron micrograph of a longitudinal section through a
bacterium (Escherichia coli); the cell's DNA is concentrated in the palely
stained region. (Courtesy of E. Kellenberger.)
vBacteria replicate quickly, dividing in two by binary
fission.
vWhen food is plentiful, "survival of the fittest"
means survival of those that can divide the fastest.
vUnder optimal conditions a single procaryotic cell
can divide every 20 minutes and thereby give rise
to 5 billion cells (approximately equal to the
present human population on earth) in less than 11
hours.
vAbility to divide quickly enables bacteria to adapt
rapidly to changes in their environment.

2 3

Family relationships between present-day More about metabolic reactions

bacteria
vBacteria live in an enormous vKEGG
variety of ecological niches, and
show a corresponding richness
in their underlying biochemical
composition.

vTwo distantly related groups

can be recognized: eubacteria,
inhabit soil, water, and larger
living organisms; and
archaebacteria, found in ocean
depths, salt brines, and hot acid
springs.

4 5
 Carbon Metabolism

6 7

Metabolic reactions are similar in all organisms The synthesis and release of oxygen into the
atmosphere
vSimilarity in all kinds of organisms, suggesting an extremely ancient
origin
vLinked to core reactions of glycolysis are hundreds of other chemical
processes
vGeneration of energy in ATP-ADP currency
vSynthesis of small molecules
vMake large polymers specific to the organism
vDegrade complex molecules, taken in as food, into simpler chemical units

8 9
Cyanobacteria were the first organisms to
The utilization of oxygen by organisms
synthesize oxygen
v As competition for raw materials for organic syntheses
intensified, a selective advantage gained by organisms able to
vExtremely reactive chemical vThe simplest of carbon molecules
utilize carbon and nitrogen atoms (in the form of CO2 and that can interact with most used by organisms is glucose
N2) directly from the atmosphere
v While they are abundantly available, it required a large
cytoplasmic constituents; must vIn the absence of oxygen glucose
amount of energy to convert CO2 and N2 to a usable organic have been toxic to many early broken down only to lactic acid or
form like simple sugars
organisms ethanol, the end products of
v The major mechanism that evolved to achieve this was
photosynthesis: radiant energy captured from the sun anaerobic glycolysis.
converted CO2 into organic compounds
v Interaction of sunlight with chlorophyll, excites an electron to
vIn the presence of oxygen glucose
a more highly energized state. As the electron drops back to a completely degraded to CO2 and
lower energy level, the energy it gives up drives chemical H2O; much more energy can be
reactions that are facilitated and directed by protein
molecules derived from each gram of glucose

10 11

Procaryotes and Eucaryotes The creation of the eukaryote cell

12 13
 The mitochondria
vOften resemble bacteria in size and shape,
vContain DNA, make protein, reproduce by
dividing in two and is responsible for
respiration.
vMany present-day bacteria respire like
mitochondria
vThe amoeba Pelomyxa palustris, while lacking
mitochondria, nevertheless carries out
oxidative metabolism by harboring aerobic
bacteria in its cytoplasm in a permanent
symbiotic relationship
14
More organelles of the eukaryote cell
16
Implications of the creation of the
mitochondria
vAcquisition of mitochondria must have had many repercussions!
vPlasma membrane is heavily committed to energy metabolism in procaryotic cells but
not in eucaryotic cells, where this crucial function has been relegated to the
mitochondria
vSeparation of functions left the eucaryotic plasma membrane free to evolve important new
features.
vBecause eucaryotic cells need not maintain a large H+ gradient across their plasma
membrane, as required for ATP production in procaryotes, it became possible to control
changes in ion permeability of the plasma membrane for cell-signaling.
vA variety of ion channels appeared in the eucaryotic plasma membrane.
vToday, these channels mediate elaborate electrical signaling processes in higher organisms -
notably in the nervous system -and they control much of the behavior of single-celled free-living
eucaryotes such as protozoa.
15
Postulated origin of the eukaryotic cell
17
Complexity of the protest – a single-celled
The length-scale of protists
creature
vThe complexity that can be
achieved by a single eucaryotic cell
is nowhere better illustrated than
in the free-living, single-celled
eucaryotes known as protists
vEvolutionarily diverse
vExhibit a bewildering variety of
different forms and
vBehaviors: photosynthetic,
carnivorous, motile, sedentary
vAnatomy: complex and includes
structures as: sensory bristles
vphotoreceptors, flagella, leg-like
appendages, mouth parts, stinging These drawings are done to different scales, but in each case the bar denotes 10
darts, muscle like contractile bundles mm. The organisms in (A), (B), (E), (F), and (I) are ciliates; (C) is an
https://www.youtube.com/watch?v=0-6dzU4gOJo euglenoid; (D) is an amoeba; (G) is a dinoflagellate; (H) is a heliozoan.
From M.A. Sleigh, The Biology of Protozoa. London: Edward Arnold, 1973.
18 19

Summary
1. Order and self-organization
a) Physical, chemical, small molecules with structural information
2.

3.
Association certain molecules according
a) RNA and proteins, later DNA
Isolation of these specialized molecules from the environment
Cellular Assemblies
a)
b)
LUCA
The central dogma DNAà RNAà Proteins
Lecture 5
4. Development of metabolic reactions to utilize specific nutrient
molecules Acknowledgements:
5. Oxygen synthesizing cells to oxygen utilizing cells Alberts - Molecular Biology of the Cell
Scitable by Nature Education
6. Procaryotes to eucaryotes
Nature Resources
7. Unicellular organisms Internet resources

Acknowledgements:
Molecular Biology of the Cell. Fifth Edition. 2007. Bruce Alberts, Alexander
Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter
20 1
 OBJECTIVE OF THE LECTURE Definitions of Self Assembly & Self Organization
o Self organization is a process in which pattern
1. Understand the meaning of self-assembly at the global level of the system emerges solely
from the numerous interactions among the
a) Some examples of artificially created self assembled structures lower-level components of the system.
2. Self assembled structures in cells Moreover, the rules specifying interactions
among the system’s components are executed
3. PDB structure using only local information without reference
to the global pattern … Camazine et al, 2003
4. Understand the principles Protein folding
ON THE OTHER HAND
o Self-assembly is the fundamental principle Flocks of Starlings
which generates structural organization on all
scales from molecules to galaxies. It is defined
as reversible processes in which pre-existing
parts or disordered components of a
preexisting system, form structures or
patterns
2 3

Examples of Self assembly Examples of Self Organization

A. Crystal structure of a ribosome.
B. Self-assembled peptide-amphiphile
nanofibers.
C. An array of millimeter-sized polymeric
plates assembled at a
water/perfluorodecalin interface by
capillary interactions.
D. Thin film of a nematic liquid crystal on an
isotropic substrate.
E. Micrometer-sized metallic polyhedra folded
from planar substrates.
F. A three-dimensional aggregate of
micrometer plates assembled by capillary
forces.
Whitesides et al, Science, 2002, 295, 2418-2421
4 5
A.An optical micrograph of a cell with fluorescently labeled
cytoskeleton and nucleus; microtubules (~24 nm in
diameter) are colored red.
B.Reaction-diffusion waves in a Belousov-Zabatinski
reaction in a 3.5-inch Petri dish.
C.A simple aggregate of three millimeter-sized, rotating,
magnetized disks interacting with one another via
vortex-vortex interactions.
D.A school of fish.
E.Concentric rings formed by charged metallic beads 1 mm
in diameter rolling in circular paths on a dielectric
support.
F.Convection cells formed above a micropatterned metallic
support. The distance between the centers of the cells is
~2 mm.
Whitesides et al, Science, 2002, 295, 2418-2421
 Self-Assembly of Mesoscopic
Metal-Polymer Amphiphiles Self assembly of snowflakes
Self assembly of gold-
polypyrole rods
vAu block diameter was 400
( 30) nm and the
polypyrrole block diameter
was 360 ( 25) nm. These
structures self-organize into
mesoscopic architectures
with unusual structures,
Snowflakes BBC
including bundles, tubes of
varying diameters, and
Hard hydrophilic domain is an inorganic material such as gold, sheets
and the soft domain is a hydrophobic conducting polymer such
as oxidized polypyrrole, which can be electrochemically Science 303, 348 (2004);
polymerized within the confines of an alumina template Sungho Park et al.6 7

Bilayer Self Assembly Tubulin and Microtubules

self assembly video

8 9
Self Assembly and self organization of internal
Images from the RCSB site
cellular structures Autophagy Cytoskeleton
Cellular Components
i. Nucleus
ii. Golgi complex
iii. Actin filaments for cellular CYTOSKETELON
structure
iv. Autophagosomes
v. Vesicles

https://ccsb.scripps.edu/goodsell/
10

Protein structure and function

vProteins can assume an unlimited number Protein Folding –
of configurations and yet possess a specific
chemical and structural function Secondary Structures
vThe known three-dimensional structures Lecture 6
of proteins are archived in the Protein
Data Bank, or PDB (www.rcsb.org/pdb) Structure of the enzyme chymotrypsin
Acknowledgements:
vThe data from the PDB files provide only a Alberts - Molecular Biology of the Cell
series of coordinates detailing the location Scitable by Nature Education
of atoms and their connectivity Nature Resources
Internet resources

12 1
Myoglobin in a Whale Muscle Cell
OBJECTIVE OF THE LECTURE 3-D structure of Proteins

1. Understand the principles Protein folding 1. the three-dimensional structure of a protein is determined by its
2. Phi-psi angles amino acid sequence
3. Ramachandran plots 2. the function of a protein depends on its structure
4. Secondary Protein structures 3. an isolated protein usually exists in one or a small number of stable
structural forms
4. the most important forces stabilizing the specific structures
maintained by a given protein are noncovalent interactions

2 3

Stability of protein structures The phi and psi angles

vThe stability of protein structures depends on weak interactions

vIt requires 200-460 kJ/mol to break a single covalent bond, compared
to 4-30kJ/mol for weak interactions
vThe weak interactions predominate because they are numerous
vThe free energies of the folded and un-folded states are similar
Governing Equations
𝝙G = 𝝙 H – 𝝙TS
𝝙G = – RTlnk
vThree bonds separate sequential a carbons in a polypeptide chain. The N—Ca and C—C a
bonds can rotate, with bond angles designated f and y, respectively
vThe peptide C—N bond is not free to rotate
vOther single bonds in the backbone may also be rotationally hindered, depending on the size
and charge of the R groups. In the conformation shown, f and y are 180 deg (or – 180 deg).
vAs one looks out from the a-carbon, the f and y angles increase as the carbonyl or amide
nitrogens (respectively) rotate clockwise

4 5
 The zero phi & psi angles The Ramachandran Plot for L-Ala
vConformations deemed possible are those that
vBy convention, both 𝛟 and 𝛙 are involve little or no steric interference, based on
defined as 0 deg when the two peptide calculations using known van der Waals radii and
bond angles.
bonds flanking that carbon are in the
vThe areas shaded dark blue reflect
same plane and positioned as shown. conformations that involve no steric overlap
and thus are fully allowed
vIn a protein this conformation is
vmedium blue indicates conformations allowed
prohibited by steric overlap between an - at the extreme limits for unfavorable atomic
carbonyl oxygen and an –amino contacts
hydrogen atom vthe lightest blue area reflects conformations
that are permissible if a little flexibility is
vTo illustrate the bonds between atoms, allowed in the bond angles
the balls representing each atom are vThe asymmetry of the plot results from the L
stereochemistry of the amino acid residues
smaller than the van der Waals radii for
this scale. 1 Å = 0.1 nm. 6 7

About Ramachandran Plots Structure of Amino Acids

1. What kind of plot do you expect for other un-branched amino-

acids?
2. What kind of plot do you expect for branched amino-acids (eg Ile)?
3. What kind of plot do you expect for glycine?
4. What kind of plot do you expect for Proline?

8 9
What are the possible applications of the
PROTEIN STRUCTURE
RAMACHANDRAN PLOT?
Overview of Protein Structure Classification of Strutures
PROTEIN STRUCTURE PREDICTION vEvery protein has a three-dimensional vSecondary
structure that reflects its function. vAlpha Helix
vProtein structure is stabilized by multiple vBeta sheets
weak interactions. Hydrophobic interactions
ALPHA FOLD are the major contributors to stabilizing the v3-D conformations
globular form of most soluble proteins;
vStructure and function
https://alphafold.ebi.ac.uk/ hydrogen bonds and ionic interactions are
optimized in the specific structures that are
thermodynamically most stable. vTertiary Structures
vThe nature of the covalent bonds in the vGlobular proteins
polypeptide backbone places constraints on
SCFBio IIT Delhi structure. The peptide bond has a partial
vMultimerics
double bond character that keeps the entire vHomo
http://www.scfbio-iitd.res.in six-atom peptide group in a rigid planar vhetero
configuration. The N–C𝜶  and C𝜶–C bonds can
rotate to assume bond angles of  𝝓 and  𝝍,
respectively.
10
11

Protein Secondary Structure

vThe a-helix architecture
vLinus Pauling, Robert Corey
vX-ray results of William Astbury (1930) of proteins that make up
porcupine quills (a-keratin)
vRegular structure that repeats every 5.16 – 5.2 Å
vpolypeptide backbone is tightly wound around an imaginary axis drawn
longitudinally through the middle of the helix, and the R groups of the a) Formation of a right-handed a-helix. The planes of the rigid peptide bonds are parallel to the long axis
amino acid residues protrude outward from the helical backbone of the helix, depicted here as a vertical rod
vThe amino acid residues in an b) Ball-and-stick model of a right-handed a- helix, showing the intrachain hydrogen bonds. The repeat
unit is a single turn of the helix, 3.6 residues
v helix have conformations with y= -45 to -50 deg and f = - 60 deg c) The a- helix as viewed from one end, looking down the longitudinal axis
d) Atoms in the center of the a- helix are in very close contact

12 13
 Amino Acid Sequence Affects a-Helix Stability
Helix Dipole

v If a polypeptide chain has a long block of Glu residues, this
segment of the chain will not form an helix at pH 7.0.
v The negatively charged carboxyl groups of adjacent Glu
residues repel each other so strongly that they prevent
formation of the a- helix
v If there are many adjacent Lys and/or Arg residues,
which have positively charged R groups at pH 7.0, they
will also repel each other and prevent formation of the
a- helix
v The bulk and shape of Asn, Ser, Thr, and Cys residues
can also destabilize an helix if they are close together in
the chain
v The twist of an helix ensures that critical interactions
occur between an amino acid side chain and the side
chain three (and sometimes four) residues away on
either side of it. Positively charged amino acids are
often found three residues away from negatively
charged amino acids, permitting the formation of an ion
pair
v Interactions between R groups of
vThe electric dipole of a peptide bond is
amino acids three residues apart in transmitted along an a-helical segment through
an a-helix
v An ionic interaction between Asp100
the intra-chain hydrogen bonds, resulting in an
and Arg103 in an a- helical region of overall helix dipole
the protein troponin C, a calcium
binding protein associated with
muscle
vThe amino and carbonyl constituents of each
v Polypeptide backbone (carbons, a- peptide bond are indicated by + and - symbols
amino nitrogens, and a-carbonyl
oxygens) is shown in gray for a helix vNon-hydrogen bonded amino and carbonyl
segment 13 residues long
constituents in the peptide bonds near each
v The interacting Asp (red) and Arg
(blue) side chains end of the a-helical region are shown in red.
14 15

Constraints affecting stability of a-helix

Antiparallel b-Sheet Conformation
1. The electrostatic repulsion (or attraction) between
successive amino acid residues with charged R groups
2. The bulkiness of adjacent R groups
3. The interactions between R groups spaced three (or four)
residues apart
4. The occurrence of Pro and Gly residues
5. The interaction between amino acid residues at the ends of
the helical segment and the electric dipole inherent to the
helix

16 17
 b-Sheet Conformation
Parallet b-Sheet Conformation
vThe zigzag polypeptide chains can be arranged side by side to form a
structure resembling a series of pleats
vHydrogen bonds are formed between adjacent segments of
polypeptide chain
vThe adjacent polypeptide chains in a sheet can be either parallel or
antiparallel (having the same or opposite amino-to-carboxyl
orientations)
vThe repeat period is shorter for the parallel conformation 6.5 Å,
versus 7 Å for antiparallel

18 19

b-turns in protein architecture

Beta Turn
v Turns that connect the ends of two adjacent segments of an antiparallel sheet.
The structure is a 180 deg turn involving four amino acid residues, with the
carbonyl oxygen of the first residue forming a hydrogen bond with the amino-
group hydrogen of the fourth

Bonding between the 1st and 3rd amino acid.

The 3rd position is usually occupied by glycine
or proline. In such positions, proline takes on a
cis-orientation (which has only about 6%
20
occurrence in proteins) 21
 OBJECTIVE OF THE LECTURE
Protein Folding – 1. 3-D conformation – structure and function
Tertiary and Quarternary Structures 2. Tertiary structures and fold symmetry
Lecture 7 3. Protein Denaturation and re-folding
4. Thermodynamic of Protein Folding
5. Chaperone Proteins
Acknowledgements:
Alberts - Molecular Biology of the Cell
Scitable by Nature Education
Nature Resources
Internet resources

1 2

3-D structure of Proteins Protein Structure of Beta Sheets and Alpha

Helices
1. the three-dimensional structure of a protein is determined by its
amino acid sequence
2. the function of a protein depends on its structure
3. an isolated protein usually exists in one or a small number of stable
structural forms
4. the most important forces stabilizing the specific structures
maintained by a given protein are noncovalent interactions

3 4
Greek Key Motif Secondary Protein Structure
vSecondary structure is the regular arrangement of amino acid
residues in a segment of a polypeptide chain, in which each residue is
spatially related to its neighbors in the same way
vThe most common secondary structures are the alpha helix, the beta
conformation, and beta turns
vThe secondary structure of a polypeptide segment can be completely
defined if the phi and psi angles are known for all amino acid
residues in that segment

5 6

vAll the amino acid residues except Gly in the enzyme pyruvate kinase (isolated
from rabbit) are overlaid on the plot of theoretically allowed conformations. The
vThe values of f and y for various allowed secondary structures are overlaid small, flexible Gly residues were excluded because they frequently fall outside
vAlthough left-handed helices extending over several amino acid residues are the expected ranges
theoretically possible, they have not been observed in proteins

7 8
 Tertiary and Quaternary Structures
vThe overall three-dimensional arrangement of all atoms in a protein
is referred to as the protein’s tertiary structure
vSome proteins contain two or more separate polypeptide chains, or
subunits, which may be identical or different. The arrangement of
these protein subunits in three-dimensional complexes constitutes
quaternary Structure
vIn considering these higher levels of structure, it is useful to classify proteins
into two major groups:
vfibrous proteins, having polypeptide chains arranged in long strands or
sheets, and
vglobular proteins, having polypeptide chains folded into a spherical or
globular shape.
9 10
Fibrous Proteins: a-keratin, collagen
vCollagen: has evolved to provide strength. It is found in
connective tissue such as tendons, cartilage, the organic
matrix of bone, and the cornea of the eye
vCollagen is also a coiled coil, but one with distinct tertiary
and quaternary structures: three separate polypeptides,
called a-chains (not to be confused with a-helices), are
supertwisted about each other
vThe tight wrapping of the chains in the collagen triple a-
helix provides tensile strength greater than that of a steel
wire of equal cross-section

a) The repeating tripeptide sequence Gly–X–

Pro or Gly–X–4-Hyp adopts a left-handed
helical structure with three residues per
turn. The repeating sequence used to
generate this model is Gly–Pro–4-Hyp
Structure of Collagen
b) Space-filling model of the same chain
c) Three of these helices (shown here in gray,
blue, and purple) wrap around one another
with a right-handed twist
d) The three-stranded collagen superhelix
shown from one end, in a ball-and-stick
representation. Gly residues are shown in
red

11 12
Collagen arrangement in a fish scale Globular Proteins

vFolding generates a compact form relative to polypeptides

in a fully extended conformation
vThe folding also provides the structural diversity necessary
for proteins to carry out a wide array of biological functions
vGlobular proteins include enzymes, transport proteins,
motor proteins, regulatory proteins, immunoglobulins, and
proteins with many other functions.

13 14

Globular Protein Structure - Myoglobin Different visualizations of Myoglobin

vThe first breakthrough in understanding the three-dimensional
structure of a globular protein came from x-ray diffraction
studies of myoglobin carried out by John Kendrew and his
colleagues in the 1950s
vMyoglobin contains a single polypeptide chain of 153 amino
acid residues of known sequence and a single iron Tertiary structure of sperm whale myoglobin
protoporphyrin, or heme, group v A ribbon representation, including side chains (blue) for the hydrophobic residues
vThe same heme group is found in hemoglobin, the oxygen-binding Leu, Ile, Val and Phe
protein of erythrocytes, and is responsible for the deep red-brown v A surface contour image is useful for visualizing pockets in the protein where other
molecules might bind
color of both myoglobin and hemoglobin.
v A space-filling model with all amino acid side chains. Each atom is represented by a
sphere encompassing its van der Waals radius. The hydrophobic residues are again
shown in blue; most are not visible, because they are buried in the interior of the16
15
protein
Common Structural Patterns
vSupersecondary structures, also called
motifs or simply folds, are particularly stable
arrangements of several elements of
secondary structure and the connections
between them
vA single large motif may comprise the entire
protein
vcoil of –keratin
vMotifs of different types occur based on
structural constraints
Rules for Folding
1. Hydrophobic interactions make a large contribution to the stability
of protein structures. Burial of hydrophobic amino acid R groups so
as to exclude water requires at least two layers of secondary
structure. Two simple motifs, the b-a-b loop and the a-a corner,
create two layers.
2. Where they occur together in proteins, a-helices and b-sheets
generally are found in different structural layers. This is because the
backbone of a polypeptide segment in the b conformation cannot
readily hydrogen-bond to an a-helix aligned with it

17 18

3. Polypeptide segments adjacent to each other in the primary a-Helix and b-Sheet content in different proteins
sequence are usually stacked adjacent to each other in the
folded structure. Although distant segments of a polypeptide vary
may come together in the tertiary structure, this is not the
norm.
4. Connections between elements of secondary structure
cannot cross or form knots
5. The b conformation is most stable when the individual
segments are twisted slightly in a right handed sense. This
influences both the arrangement of b sheets relative to one
another and the path of the polypeptide connection between
them.

19 20
Structural Classification of Proteins
vThe Structural Classification of Proteins (SCOP) database
offers a good example of this very important trend in
biochemistry
vAt the highest level of classification, the SCOP database
(http://scop.mrc-lmb.cam.ac.uk/scop) borrows a scheme already
in common use
vall a
vall b
va/b (in which the a and b segments are interspersed or
alternate)
va and b (in which the and regions are somewhat segregated)
21 22

Quarternary Protein Structures Hemoglobin

vMany proteins have multiple polypeptide subunits. The association
of polypeptide chains can serve a variety of functions. vX-ray diffraction analysis of
vMany multisubunit proteins have regulatory roles
deoxyhemoglobin (hemoglobin without
oxygen molecules bound to the heme
vThe binding of small molecules may affect the interaction between groups) shows how the four polypeptide
subunits, causing large changes in the protein’s activity in response to subunits are packed together
small changes in the concentration of substrate or regulatory
molecules v A ribbon representation.
vA multisubunit protein is also referred to as a multimer vA space-filling model. The a subunits are
shown in gray and light blue; the b subunits
vFew subunits – Oligomers in pink and dark blue
vRepeating subunits - protomers vthe heme groups (red) are relatively far apart

23 24
 Cyclic Symmetry

vIdentical subunits of multimeric proteins are generally

arranged in one or a limited set of symmetric patterns. A
description of the structure of these proteins requires an
understanding of conventions used to define symmetries.
vOligomers can have either rotational symmetry or helical
symmetry
vIn cyclic symmetry, subunits are related by rotation about a
single n-fold axis, where n is the number of subunits so
related. The axes are shown as black lines; the numbers are
values of n. Only two of many possible Cn arrangements are
shown
25 26

Dihedral Symmetry Icosahedral Symmetry

vIcosahedral symmetry. Relating all 20
triangular faces of an icosahedron
requires rotation about one or more of
three separate rotational axes: twofold,
threefold, and fivefold.

vIn dihedral symmetry, all subunits can be related by

rotation about one or both of two axes, one of which is
twofold. D2 symmetry is most common.

27 28
 Tertiary and Quarternary Structure Denaturation and folding
vTertiary structure is the complete 3-D structure of a polypeptide chain. There are two
general classes of proteins based on tertiary structure: fibrous and globular. vProteins are marginally stable
vFibrous proteins, which serve mainly structural roles, have simple repeating elements vChanges in the environmental conditions affects structure and function
of secondary structure.
vDenaturation is the process in which change in the 3-D structure of
vGlobular proteins have more complicated tertiary structures, often containing several the protein is sufficient to cause a loss of function
types of secondary structure in the same polypeptide chain. The first globular protein
structure to be determined, using x-ray diffraction methods, was that of myoglobin. vFactors:
vThe complex structures of globular proteins can be analyzed by examining stable vTemperature
substructures called supersecondary structures motifs, or folds. The thousands of vpH
known protein structures are generally assembled from a repertoire of only a few
hundred motifs. Regions of a polypeptide chain that can fold stably and vSolvents (acetone, alcohol)
independently are called domains. vSolutes such as urea, guanidine hydrochloride
vQuaternary structure results from interactions between the subunits of multisubunit vDetergents
(multimeric) proteins or large protein assemblies. Some multimeric proteins have a
repeated unit consisting of a single subunit or a group of subunits referred to as a
protomer. Protomers are usually related by rotational or helical symmetry
29 30

Renaturation of
Protein Denaturation unfolded Protein

vUrea is used to denature

ribonuclease, and mercaptoethanol
(HOCH2CH2SH) to reduce and thus
cleave the disulfide bonds to yield
• Denaturation (a) by temperature change (b) by addition of guanidine HCl eight Cys residues. Renaturation
monitored using Circular Dichroism involves reestablishment of the
correct disulfide cross-links.

31 32
Protein folding simulation
vThe process started with the randomly coiled peptide and 3,000
surrounding water molecules in a virtual “water box.” The molecular
motions of the peptide and the effects of the water molecules were
taken into account in mapping the most likely paths to the final
structure among the countless alternatives.
33
GroEL-GroES – member of HSP 60 family
Chaprones in Protein Folding
35
Thermodynamics of Protein Folding
vThe number of conformations, and hence the
conformational entropy, is large. Only a small
fraction of the intramolecular interactions
that will exist in the native conformation are
present.
vAs folding progresses, the thermodynamic
path down the funnel reduces the number of
states present (decreases entropy), increases
the amount of protein in the native
conformation, and decreases the free energy.
vDepressions on the sides of the funnel
represent semistable folding intermediates,
which may, in some cases, slow the folding
process.
34
36
 Objectives of the lecture
1. Learn about the background research that led to the deciphering of
the Genetic Code
Lectures 9 2. The Experiment of Nirenberg
3. The establishment of the genetic code
The Genetic Code

Acknowledgements:
Leninger Chap 27
Scitable
Internet Resources

Important Contributions that Lead to the

The protein synthesis process is complex
Deciphering of the Genetic Code
1. Eukaryotic protein synthesis involves more than
a) 70 different ribosomal proteins
b) 20 or more enzymes to activate the amino acid precursors
c) a dozen or more auxiliary enzymes and other protein factors for the initiation,
elongation, and termination of polypeptides
d) 100 additional enzymes for the final processing of different protein
e) 40 or more kinds of transfer and ribosomal RNAs
2. Overall, almost 300 different macromolecules cooperate to synthesize
Gregor Mendel Walter Sutton Thomas Hunt Morgan
polypeptides
3. Every procaryote or eucaryote cell has thousands of copies of different o Elementen
RNAs and proteins which constitutes about 35% of the cell dry weight Existence of
o External Chromosome
4. Protein synthesis utilizes about 90% of the chemical energy of the cell resemblance s in pairs
o Internal nature
Deciphering the Genetic Code
v1865 –Mendel defined the basic unit of inheritance as the gene
George Gamow and the “RNA tie Club”
v1900 –Mendel’s forgotten work resurfaces; nature of gene is still
unknown vBrotherhood consisted of 20 regular members,
one for each amino acid
v1944 –it is established that a gene is made of DNA vWatson was PRO (proline)
v1953 –Watson-Crick’s double helix structure for DNA vFour honorary members, one for each
DNA: L = {A, C, G, T} nucleotide
One big question
RNA: L = {A, C, G, U} vEight of these members were or became Nobel
remained unanswered:
Double Stranded DNA Laureates Georgiy Antonovich Gamov
how is the information March 4, 1904- August 19, 1968
5’ A T T G C C C A T 3’ Big Bang Theory
in the DNA strand Formation of stars

3` T A A C G G G T A 5’ translated to protein?

Some of the Ideas Proposed Combinatorial Figures of Gamow’s

by the RNA Club proposition

vThe Adapter Hypothesis by Francis Crick – some

unknown biological entity carried the amino acids and
put them in the sequence order
vGamow proposed that a three-letter code would be vNumber of diamonds where top and
sufficient to define all 20 amino acids bottom are identical
v4C1×2 =8
vNumber of diamonds where top and
bottom are different
v4C2×2 =12
 Marshall Nirenberg Deciphered
Analysis Presented by Crick
vGenetic code is in triplets (codon) the Genetic Code in 1961
vThere are 20 amino acids
vThere are 4 alphabets A, T, G, C
v4 & 42< 20, ∴43 = 64 (but with redundancies ?)
vThe genetic code should be comma free
Marshall Warren Nirenberg
vOnly one valid reading frame April 10, 1927 – January 15,
v[abc][def][ghi][jkl] 2010, ; Jewish American
vNOT a [bcd][efg]hij]… biochemist and geneticist
vNOT ab[cde][fgh][ijk]…

(AND THE FOLLOWING THOUGH NOT QUITE CORRECT)

vAAA, TTT, GGG, CCC are not possible because for example AAAAAA the reading frame
is ambiguous
vThat leaveS 64-4 = 60
vATGATG must be read unambiguously, So, whenever ATG is a codon, TGA or GAT is not
M. W. Nirenberg and P. Leder, 1964, Science 145:1399
vThat gives (1/3)*60 = 20

Grouping by Physical Properties of Amino

The Genetic Code
Acids Best Explains the Genetic Code Table

The genetic
code is a map of
Codons “C” to
Amino Acids “A”
g: C à A
 Degeneracy of the Genetic Code
Important points related to translation
vThe particular amino acid sequence of a protein is constructed through the
translation of information encoded in mRNA. This process is carried out by
ribosomes.
vAmino acids are specified by mRNA codons consisting of nucleotide
triplets. Translation requires adaptor molecules, the tRNAs, that recognize
codons and insert amino acids into their appropriate sequential positions
in the polypeptide.
vThe base sequences of the codons were deduced from experiments using
synthetic mRNAs of known composition and sequence.
v The codon AUG signals initiation of translation. The triplets UAA, UAG,
and UGA are signals for termination.

The Wobble Hypothesis

vAlignment of the two RNAs is
antiparallel. The tRNA is shown in
the traditional cloverleaf
configuration
vThree different codon pairing
relationships are possible when the
tRNA anticodon contains inosinate.
Reading Frames
The Triplet Non-overlapping Code

Objectives of this Lecture

1. Stages of Protein Synthesis
2. The role of ribosomes
Lecture 10 3. The role of tRNA
4. Details of each of the stages of protein synthesis
Protein Synthesis

Acknowledgements:
Leninger Chap 27
Scitable
Internet Resources
 Ribosome is a Complex Supramolecular
Five Stage of Protein Synthesis in E. coli
Machine
1. Activation of Amino Acids 1. Each E. coli contains 15000 or more ribosomes (1/4 cell weight)
2. Initiation 2. 18 nm is size
3. Elongation 3. Two subunits (i) 30S (ii) 50S combined 70S (S is the sedimentation
4. Termination coefficient)
5. Folding and post translational processing 4. Subunits are made of many proteins and at least one large rRNA
5. Bacterial ribosomes have 55 proteins with molecular weights
varying from 6000 to 75000
Look up: PDB ID 1JJ2 and 1GIY

The Ribosome
Bacterial rRNA
Secondary structure
of E. coli 16S and 5S
rRNAs. The first (5’
end) and final (3’
end) ribonucleotide
residues of the 16S
rRNA are
numbered.
 Aminoacyl t-RNA Synthetase
Nobel Prize in Chemistry 2009
Aminoacyl-tRNA Synthetases
The Nobel Prize in Chemistry 2009 Attach the Correct Amino Acids to
Their tRNAs
was awarded jointly to Venkatraman vaminoacyl-tRNA synthetases
Ramakrishnan, Thomas A. Steitz and esterify the 20 amino acids to their
corresponding tRNAs. Each
Ada E. Yonath "for studies of the enzyme is specific for one amino
acid and one or more
structure and function of the corresponding tRNAs
ribosome" v Proofreading by Aminoacyl-tRNA
Synthetases
v Interaction between an
Visit to KSBS Aug 2020 Aminoacyl-tRNA Synthetase and a
tRNA
https://www.nobelprize.org/prizes/chemistry/2009/summary/

2-D and 3-D structure of tRNA Stage 1: Attaching the correct amino acid to
the correct tRNA
in the cytosol, aminoacyl-tRNA synthetases esterify the 20
amino acids to their corresponding tRNAs.
v Each enzyme is specific for one amino acid and one or
more corresponding tRNAs
v Most organisms have one aminoacyl-tRNA synthetase
for each amino acid
v For amino acids with two or more corresponding
tRNAs, the same enzyme usually aminoacylates all of
them
TWISTED “L” STRUCTURE
 Proof Reading by Aminoacyl tRNA
Synthetases
Aminoacylation of tRNA
accomplishes
1. Activation of amino acid for
peptide bond formation
2. attachment of amino acid to
an adaptor tRNA for
placement of amino acid
The amino acid attached is not
checked on the ribosome!
Formation of the
Initiation Complex
1. 30S ribosomal subunit
2. mRNA coding for the
polypeptide to be
made
3. Initiating fMet-
tRNAfMet
4. A set of three proteins
called initiation factors
(IF-1, IF-2, and IF-3)
5. GTP
6. 50S ribosomal subunit
7. Mg2+.
How is the fidelity assured?
Consider Valine and Isoleucine -
different by only – CH2 –
In the case of Ile-tRNA synthetase
1. Activation of Ile is favored by a
factor of 200
2. Binding is carried out in 2 steps
(acts as filter)
3. Incorrect binding occurs at a second
site that has a higher hydrolytic rate
4. In this case, overall process is
1:3000 in favor of the correct amino
acid Ile
Stage 2: A Specific Amino Acid Initiates Protein Synthesis
Although methionine has only one codon, (5’)AUG, all organisms have two
tRNAs for methionine
v One is used exclusively when (5’)AUG is the initiation codon for protein
synthesis
v The other is used to code for a Met residue in an internal position in a
polypeptide
v The amino acid incorporated in response to the (5’)AUG initiation codon
is N-formylmethionine (fMet)
Methionine + tRNAfMet + ATP à Met-tRNAfMet + AMP + PPi
Stage 3: Elongation
of the Peptide Chain
Elongation requires
1. the initiation complex
2. aminoacyl-tRNAs
3. a set of three soluble
cytosolic proteins called
elongation factors (EF-Tu,
EF-Ts, and in bacteria)
4. GTP
Stage 3 : Translocation
1. The ribosome moves one codon toward the 3’
end of the mRNA
This shifts the anticodon of the tRNA attached to the 2nd
codon from the AàP site and the deacylated tRNA from
Pà E site
2. This movement required EF-G (translocase)
and the energy is provided by the hydrolysis
GTPàGDP+Pi
3. The uncharged tRNA is dislocated from the E-
site and a peptide bond is formed between
the growing chain and the new amino acid
carried by the tRNA at the A-site
Summary of the
5 stages of
protein synthesis
Stage 4: Termination of
Synthesis
1. Elongation continues until the last
amino acid in the sequence
2. Termination is signaled by the presence
of one of the 3 stop codons UAA, UAG
or UGA immediately following the final
coded amino acid
3. Once the terminal codon occupies A-
site, three termination or release
factors RF1, RF2 and RF3 contribute to
i. Hydrolysis of terminal peptidyl tRNA bond
ii. Release of polypeptide from P-site
iii. Dissociation of the 70S ribosome into the
30S and 50S subunits
Lecture 12
Regulation of Gene Expression
Negative regulation - Lac Operon
Attenuation - Tryptophan Operon
Acknowledgement: Leninger Chapter 28
Objectives Genes are expressed when required
Emergence of Life 1. Understanding gene regulation vSome proteins are expressed abundantly such as elongation factors
Fundamental units of life a) Operons and regulons and rubisco
Cellular assemblies 2. Negative and positive vOthers such as DNA repair enzymes are synthesized very few in
Protein Folding regulation number
Protein Synthesis 3. Lac operon vRequirements of gene products varies in the cell-type and in its life
Gene Regulation 4. Attenuation regulation – Tryp cycle
operon vRibosomes are synthesized rapidly during the exponential growth phase of
the cell

What factors determine the cellular

concentration of proteins
1. Synthesis of the primary RNA transcript (transcription)
2. Posttranscriptional modification of mRNA
3. Messenger RNA degradation
4. Protein synthesis (translation)
5. Posttranslational modification of proteins
6. Protein targeting and transport
7. Protein degradation
Gene regulation Representative Prokaryotic Operon

House Keeping
Regulated Genes
Genes
•Constitutive gene •Inducible gene
expression expression
•Repressible gene
expression vGenes A, B, and C are transcribed on one polycistronic mRNA. Typical
regulatory sequences include binding sites for proteins that either
activate or repress transcription from the promoter

RNA polymerase
Negative Regulation of Gene Expression

vRNA polymerases bind to DNA and initiate transcription at promoters, sites

generally found near points at which RNA synthesis begins on the DNA template
vThe regulation of transcription initiation often entails changes in how RNA
polymerase interacts with a promoter
 Positive Regulation of Gene Expression Lactose metabolism in E. coli

The Lac Operon

The Lac Operon

vThe I gene encodes the Lac repressor.
The lac Z, Y, and A genes encode beta-
galactosidase, galactoside permease,
and thiogalactoside transacetylase,
respectively
vO1 is the main operator for the lac operon
vThe Lac repressor binds to the main
operator and O2 or O3, apparently
forming a loop in the DNA that might
wrap around the repressor
The Trp Operon This operon is regulated by two
mechanisms:
vWhen tryptophan levels are
high, the repressor binds to its
operator
vTranscription of trp mRNA is
attenuated

What happens at high Tryptophan levels

The Trp mRNA Sequence

v When tryptophan levels are high, the ribosome quickly translates sequence 1 (open reading frame encoding
leader peptide) and blocks sequence 2 before sequence 3 is transcribed. Continued transcription leads to
attenuation at the terminator-like attenuator structure formed by sequences 3 and 4
 What happens at low Tryptophan levels

Computational Aspects of
Gene Regulation
Lecture 13

v When tryptophan levels are low, the ribosome pauses at the Trp codons in sequence 1. Formation of the
paired structure between sequences 2 and 3 prevents attenuation, because sequence 3 is no longer
available to form the attenuator structure with sequence 4. The 2:3 structure, unlike the 3:4 attenuator, does
not prevent transcription. 1

Objectives The Lac and Trp Operons

In this lecture you will learn about:

Lac Operon Trp Operon
1. What are gene regulatory networks
2. Steps in building a gene regulatory network
3. Model of a transcription module
4. Network motifs and logic gates
5. Boolean representations
3

2
 What are Gene Regulatory (Transcription)
A typical gene regulatory network Networks?
This is one of the layers of information Signal
generation and transmission within a molecules

cell
vIt is the regulation of gene expression at the Gene
stage of transcription
vThis is broadly the first step to gene expression RNA
and its control regulations the temporal
programming in genes
Proteins
vIt is of interest because it mediates changes in
cells and helps in understanding the onset and
progression of disease substrates products
4 5

There are
Regulatory different
Network of methods of
E. coli K12 analysis

6 7
Steps to follow for building a regulatory Add the details of the important constituents
network model
vIdentify the elements of the model
vCharacterize the kind of
interaction/reaction
vDefine the boundary of your observation
(system)
vIdentify the information/flow “into” and
“out of” the boundary
vWhat are the intrinsic vLayout the reactions involved
generation/degradation rates?
vAre the rates balanced?
vAre all the parameters known?
vAssign the kinetics
vCode and Simulate
8 9

Performing simulation and analysis of results

Define the rate equations
vThis is the most important step !
vGo to the literature and ensure
!"
!
correctness of the reactions !"
vUse various resources to determine !
the parameters that have been !"
reported for the same or similar 𝑑 propose?
reactions (or cellular events) 𝑑𝑡
vDo your experiments results appear 𝑑 𝑑
reasonable? Have the parameters
been evaluated correctly? 𝑑𝑡
vCross check the results to make
sure that it makes sense
vCheck the boundary results
vDo the values at the boundaries
satisfy the physical constraints?
vPlot the data/results and analyze
𝐺𝑙𝑢 = 𝑣# -𝑣$
vWhat is the insight you have
𝐺6𝑃 = 𝑣$ -𝑣% gained?
𝐹6𝑃 = 𝑣% -𝑣& +𝑣'
vWhat is the hypothesis you can
𝐹1,6𝑃2 = 𝑣& −𝑣' +𝑣(
𝐴𝑇𝑃 = − 𝐴𝐷𝑃 = −𝑣$ −𝑣& +𝑣)
𝑑𝑡

10 11
 Transcription Modules Example of a cascade of Mod 1 R1 I1
Mod 2

genes
vIn the general form, the ODE that models the Regulator protein
(R) gene1 Mod 3
P
output of the gene (Z) in response to a Operator sites
Inducer (I)
R2
regulatory input S is given by the equation Z
Z vPromoter P has no regulatory P1
gene2
dZ k × ( S n / K n )µ inputs
= k¢ + - kd × Z DNA
reporter
dt 1 + (S n / K n ) GENE
vP is constitutive and drives the P2

k æ (S n / K n )µ ö Promoter (P) expression of R1 which in turn

Z ss = çç a + ÷, k ¢ = a × k
kd è 1 + ( S n / K n ) ÷ø inhibits R2 dR2 k × ( I / K ) n1
vThe inducer I1 regulates the = a1k1 + 1 1 1 n1 - kd 2 × R2

Fluorescence (gfp)
vRepression an Activation are taken care of by dt 1 + ( I1 / K1 )
signal R2/P2 by modulating the dZ k2
the parameter µ cellular abundance of R2 = a2 k 2 + - kd × Z
dt 1 + ( R2 / K 2 ) n2
µ = 0 à repression; µ = 1 à activation
k1 æ k × ( I / K )n1 ö
vThe parameters k’ and k, represent the signal- R2 ss = çç a1 + 1 1 1 n1 ÷÷
P1: n = 2.4, K = 5.5 nM, k = 220 min-1, kd 2 è 1 + ( I1 / K1 ) ø
independent and the signal-dependent gene Inducer Concentration
k2 æ k2 ö
expression. P2: n = 1.7, K = 120 nM, k = 255 min-1, Z ss = çç a2 + ÷÷
12
kd è 1 + ( R2 ss / K 2 )n2 ø 13

Oscillatory Networks – The Repressilator Regulatory circuits can also be represented as graphs
R2 R3
Fluorescence

P1
gene2 gene3 gene1
P2 P3
v1

-1
R1 time
-1
vRepressor R1 inhibits the expression of
v3
repressor R2, repressor R2 inhibits the
dmi k -1
expression of repressor R3, and repressor R3 = ak + - mi v2
inhibits the expression of repressor R1 dt 1 + rjn
vThe separation of transcription and
translation contributes to a response delay dri
that results in the emergence of oscillations = e (mi - ri )
dt
14 15
 Gene Regulation can also be treated as logic gates
Signed Graphs
vArabinose is only used if glucose is not present; proteins in
this system are made only when condition arabinose “AND
NOT” glucose is satisfied
A signed graph S is an undirected vThe delay TON ~20 min
network whose edges have functional v1
vX = CRP, SX = cAMP, Sy = arabinose, Y = araC,
In the lactose operon, X does not regulate Y = lacI
values of +1 or –1 ; it is natural to refer
to them as a positive edge or negative +1 -1

edge.
v2 -1 v3
For example:
V = (v1, v2, v3), E = (v1v2, v2v3, v3v1) and
f ={(v1v2,+1) (v2v3, –1) (v3v1, –1) }
16 J. Mol Biol , 356, 1073. 17

Structure of the feed-forward loop The FFL is a persistence detector

vThe feedforward loop in consideration vA sign sensitive delay element can be
vHas a direct path from X to Z considered as an asymmetric filter
v has an indirect path X to Y to Z vA brief pulse of X is results in a signal
vEach edge can be an activation or repression; so shorter that TON
there are 23=8 FFLs
vHowever, the motif responds
vThese are classified into two groups
immediately to a pulse OFF signal
vCoherent: the indirect path has the same overall sign
as the direct path
vIncoherent: the sign of the indirect path is opposite to
that of the direct path
vThere are two possible logic gates for the expression of Z :
AND or OR Sign sensitive delays protect the gene circuit
– the synthesis of Z is not initiated until the
signal is confirmed – thereby energy is
conserved
18 19
 Yeast cell cycle Boolean Attractors
vA boolean network is defined by G(V, F)

vLet vi(t) represent the state of vi at time t. The overall

expression level of all the genes in the network at time step t
is given by the following vector:

vThere are 2n possible states; the regulatory rules among the

genes are given as follow
Gene network for
Yeast cell cycle
Temporal evolution of states
Li F et al. PNAS 2004;101:4781-4786
20 21

Developing New Methods of Analysis – An Example

Toy Network Example v2
v1 v2

v3 v3
Recruit important networks for building the global v1 v2
network v4
v2
• Network analysis v5 v3
v5
• Observability-Controllability analysisN k

i
å
< 2 N to
• Boolean – for large networks where it 2is difficult
i v4
v4
v1

find kinetic data v5

• Faster algorithms More than 100 times faster vDivide the network into v1
• Determination of attractors
for real networks subgraphs of individual v4
nodes
v5 v3
22 23
Determine the steady states of subgraphs
v2 v2

v5 v1 v1

v1 v2 v3 v5
v4

v3 v4 v4 v5 v3

V1 V2 V3 V2 V4 V5 V3 V2 V4 V4 V1 V5 V5 V1 V3
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 0 1 0 1 0 0 1 0 0 0 1 0 1 0
1 1 0 0 1 1 0 1 1 0 1 0 0 1 1
1 1 1 1 0 0 1 0 0 0 1 1 1 0 0
1 0 1 1 0 1 1 0 0 1 0 1
1 1 1 1 1 1 1 1 0
1 1 1
24

Review
HIGHLIGHTS OF THE IMPORTANT TOPICS OF THE COURSE
Lecture 11
Vesicle of any desired size and shape can be
synthesized under lab conditions
vDepends on mixing entropies (pull towards
many assemblies) and molar bending
energies ( tends towards a smaller number
of vesicles)
vDA is the difference in area, v is the
dimensionless volume to area ratio
We discussed how principles of self-assembly
leads to the notion that it is possible that a living
cell could be created under the conditions existing
in primordial earth
In-animate Systems
vPhysical systems
vChemical systems
vBiological self-
assembled systems
Living Systems
vThe cell
1 2
Last Universal Common Ancestor
Adv Mater 2003 15 (16) vThe evolution of the
translation apparatus
occurred in a series of
increasingly complex stages,
rather than all at once,
vThe stages subsequent to the
establishment of the basic
mechanism were concerned
by and large with increasing
the mechanism's accuracy,
AFM image of a polymer and possibly speed as well
vesicle. The erythrocyte-
J. Mol Evol 10, 1-6, 1977
like shape arises from the
evaporation of water from
the vesicle interior,
leaving the prominent rim
3 4
 Procaryotic Cells Are Structurally Simple but
Fundamental Units of Life Biochemically Diverse

The main biomolecules v Bacteria are the simplest

that are part of the organisms found in most natural
building blocks of cells environments.
vCarbohydrates and v Spherical or rod-shaped cells,
Lipids – cell structure commonly several micrometers in
linear dimension Possess a tough
vAmino acids – proteins protective coat, cell wall, beneath
vNucleotides – DNA and which a plasma membrane
RNA encloses a single cytoplasmic
compartment containing DNA,
RNA, proteins, and small Procaryote sizes and structures. (A) Some procaryotic cells drawn to
scale. (B) Electron micrograph of a longitudinal section through a
molecules. bacterium (Escherichia coli); the cell's DNA is concentrated in the palely
stained region. (Courtesy of E. Kellenberger.)

5 6

More about metabolic reactions

vKEGG vSimilarity in all kinds of organisms,

suggesting an extremely ancient
origin
vLinked to core reactions of
glycolysis are hundreds of other
chemical processes
vGeneration of energy in ATP-ADP
currency
vSynthesis of small molecules
vMake large polymers specific to the
organism
vDegrade complex molecules, taken in
as food, into simpler chemical units

7 8
 Carbon Metabolism The creation of the eukaryote cell

9 10

Postulated origin of the eukaryotic cell Self Assembly and self organization of internal
cellular structures

Flocks of Starlings

Autophagy Cytoskeleton
Images from the RCSB site
https://ccsb.scripps.edu/goodsell/
11 12
Protein Folding

v Secondary Structures
vAlpha Helix
vBeta Sheet
vTertiary Structure (3-D conformation)
vQuarternary Structure
a) Formation of a right-handed a-helix. The planes of the rigid peptide bonds are parallel to the long axis
of the helix, depicted here as a vertical rod
b) Ball-and-stick model of a right-handed a- helix, showing the intrachain hydrogen bonds. The repeat
unit is a single turn of the helix, 3.6 residues
c) The a- helix as viewed from one end, looking down the longitudinal axis
d) Atoms in the center of the a- helix are in very close contact

13 14

Antiparallel b-Sheet Conformation Parallet b-Sheet Conformation

15 16
 Stability of protein structures
1. The stability of protein structures depends on weak interactions
2. It requires 200-460 kJ/mol to break a single covalent bond,
compared to 4-30kJ/mol for weak interactions
a) The weak interactions predominate because they are numerous
3. The free energies of the folded and un-folded states are similar
Governing Equations
𝝙G = 𝝙 H –T 𝝙 S
𝝙G = – RTlnk
The zero phi & psi angles
vBy convention, both 𝛟 and 𝛙 are
defined as 0 deg when the two peptide
bonds flanking that carbon are in the
same plane and positioned as shown.
vIn a protein this conformation is
prohibited by steric overlap between an -
carbonyl oxygen and an –amino
hydrogen atom
vTo illustrate the bonds between atoms,
the balls representing each atom are
smaller than the van der Waals radii for
this scale. 1 Å = 0.1 nm.
The phi and psi angles
vThree bonds separate sequential a carbons in a polypeptide chain. The N—Ca and C—C a
bonds can rotate, with bond angles designated f and y, respectively
vThe peptide C—N bond is not free to rotate
vOther single bonds in the backbone may also be rotationally hindered, depending on the size
and charge of the R groups. In the conformation shown, f and y are 180 deg (or – 180 deg).
vAs one looks out from the a-carbon, the f and y angles increase as the carbonyl or amide
nitrogens (respectively) rotate clockwise
17 18
The Ramachandran Plot for L-Ala
vConformations deemed possible are those that
involve little or no steric interference, based on
calculations using known van der Waals radii and
bond angles.
vThe areas shaded dark blue reflect
conformations that involve no steric overlap
and thus are fully allowed
vmedium blue indicates conformations allowed
at the extreme limits for unfavorable atomic
contacts
vthe lightest blue area reflects conformations
that are permissible if a little flexibility is
allowed in the bond angles
vThe asymmetry of the plot results from the L
stereochemistry of the amino acid residues
19 20
Secondary Protein Structure
vSecondary structure is the regular arrangement of amino acid
residues in a segment of a polypeptide chain, in which each residue is
spatially related to its neighbors in the same way
vThe most common secondary structures are the alpha helix, the beta
conformation, and beta turns
vThe secondary structure of a polypeptide segment can be completely
defined if the phi and psi angles are known for all amino acid
residues in that segment
vThe values of f and y for various allowed secondary structures are overlaid
vAlthough left-handed helices extending over several amino acid residues are
theoretically possible, they have not been observed in proteins

21 22

Symmetry in Tertiary Structures:

Icosahedral Symmetry

vIcosahedral symmetry. Relating all 20

triangular faces of an icosahedron
requires rotation about one or more of
three separate rotational axes: twofold,
threefold, and fivefold.
vAll the amino acid residues except Gly in the enzyme pyruvate kinase (isolated
from rabbit) are overlaid on the plot of theoretically allowed conformations. The
small, flexible Gly residues were excluded because they frequently fall outside
the expected ranges

23 24

Renaturation of
unfolded Protein
vUrea is used to denature
ribonuclease, and mercaptoethanol
(HOCH2CH2SH) to reduce and thus
cleave the disulfide bonds to yield
eight Cys residues. Renaturation
involves reestablishment of the
correct disulfide cross-links.
Thermodynamics of Protein Folding
WHAT IS THE LEVINTHAL’S
PARADOX?
2 MODELS OF FOLDING
25
1. The number of conformations,
and hence the conformational
entropy, is large. Only a small
fraction of the intramolecular
interactions that will exist in the
native conformation are present.
2. As folding progresses, the
thermodynamic path down the
funnel reduces the number of
states present (decreases
entropy), increases the amount
of protein in the native
conformation, and decreases the
free energy.
3. Depressions on the sides of the
funnel represent semistable
folding intermediates, which may,
in some cases, slow the folding
process. 27
Protein folding simulation
v The process started with the randomly coiled peptide and 3,000
surrounding water molecules in a virtual “water box.” The molecular
motions of the peptide and the effects of the water molecules were
taken into account in mapping the most likely paths to the final structure
among the countless alternatives.
v 1 ms simulation time took half a billion integration steps on 2 Cray
Computers
26
PROTEIN MISFOLDING IS THE CAUSE OF
MANY FATAL DISEASES
vSpongiform encephalopathies – brain gets riddled with holes
vCreutzfeldt-Jacob disease – fatal illness; symptoms – dementia, loss of
coordination
vStanley Prusiner traced the cause to PRION (proteinaceous infectious
only) protein (PrP)
vTwo form – normal cellular form (PrP or PrPC) and altered conformation
”scrapie” form (PrPSc)
Cells have “Chaperone” proteins to assist proteins in
folding correctly or refold proteins that have been
denatured by heat or other conditions
28
GroEL-GroES – member of HSP 60 family
The Genetic Code

The genetic
code is a map of
Codons “C” to
Amino Acids “A”
g: C à A

29

Grouping by Physical Properties of Amino

Acids Best Explains the Genetic Code Table Important points related to translation

vThe particular amino acid sequence of a protein is constructed through the

translation of information encoded in mRNA. This process is carried out by
ribosomes.
vAmino acids are specified by mRNA codons consisting of nucleotide
triplets. Translation requires adaptor molecules, the tRNAs, that recognize
codons and insert amino acids into their appropriate sequential positions
in the polypeptide.
vThe base sequences of the codons were deduced from experiments using
synthetic mRNAs of known composition and sequence.
v The codon AUG signals initiation of translation. The triplets UAA, UAG,
and UGA are signals for termination.
Degeneracy of the Genetic Code
The Wobble Hypothesis
vAlignment of the two RNAs is
antiparallel. The tRNA is shown in
the traditional cloverleaf
configuration
vThree different codon pairing
relationships are possible when the
tRNA anticodon contains inosinate.
Reading Frames
The Triplet Non-overlapping Code Five Stage of Protein Synthesis in E. coli
1. Activation of Amino Acids
2. Initiation
3. Elongation
4. Termination
5. Folding and post translational processing

Ribosome is a Complex Supramolecular

Bacterial rRNA
Machine
1. Each E. coli contains 15000 or more ribosomes (1/4 cell weight) Secondary structure
of E. coli 16S and 5S
2. 18 nm is size rRNAs. The first (5’
3. Two subunits (i) 30S (ii) 50S combined 70S (S is the sedimentation end) and final (3’
coefficient) end) ribonucleotide
4. Subunits are made of many proteins and at least one large rRNA residues of the 16S
rRNA are
5. Bacterial ribosomes have 55 proteins with molecular weights numbered.
varying from 6000 to 75000
PDB ID 1JJ2 and 1GIY
 The Ribosome
Nobel Prize in Chemistry 2009

The Nobel Prize in Chemistry 2009

was awarded jointly to Venkatraman
Ramakrishnan, Thomas A. Steitz and
Ada E. Yonath "for studies of the
structure and function of the
ribosome"

Visit to KSBS Aug 2020

https://www.nobelprize.org/prizes/chemistry/2009/summary/

Aminoacyl t-RNA Synthetase

Aminoacyl-tRNA Synthetases
Attach the Correct Amino Acids to
Their tRNAs
vaminoacyl-tRNA synthetases
esterify the 20 amino acids to their
corresponding tRNAs. Each
enzyme is specific for one amino
acid and one or more
corresponding tRNAs
v Proofreading by Aminoacyl-tRNA
Synthetases
v Interaction between an
Aminoacyl-tRNA Synthetase and a
tRNA
 Stage 1: Attaching the correct amino acid to Proof Reading by Aminoacyl tRNA
the correct tRNA Synthetases
in the cytosol, aminoacyl-tRNA synthetases esterify the 20 Aminoacylation of tRNA How is the fidelity assured?
amino acids to their corresponding tRNAs. accomplishes Consider Valine and Isoleucine -
different by only – CH2 –
v Each enzyme is specific for one amino acid and one or
more corresponding tRNAs
1. Activation of amino acid for In the case of Ile-tRNA synthetase
peptide bond formation 1. Activation of Ile is favored by a
v Most organisms have one aminoacyl-tRNA synthetase factor of 200
for each amino acid 2. attachment of amino acid to 2. Binding is carried out in 2 steps
v For amino acids with two or more corresponding an adaptor tRNA for (acts as filter)
tRNAs, the same enzyme usually aminoacylates all of placement of amino acid 3. Incorrect binding occurs at a second
them site that has a higher hydrolytic rate
The amino acid attached is not 4. In this case, overall process is
checked on the ribosome! 1:3000 in favor of the correct amino
acid Ile

Stage 2: A Specific Amino Acid Initiates Protein Synthesis Formation of the

Although methionine has only one codon, (5’)AUG, all organisms have two Initiation Complex
tRNAs for methionine 1. 30S ribosomal subunit
v One is used exclusively when (5’)AUG is the initiation codon for protein 2. mRNA coding for the
synthesis polypeptide to be
v The other is used to code for a Met residue in an internal position in a made
polypeptide 3. Initiating fMet-
v The amino acid incorporated in response to the (5’)AUG initiation codon tRNAfMet
is N-formylmethionine (fMet)
4. A set of three proteins
called initiation factors
Methionine + tRNAfMet + ATP à Met-tRNAfMet + AMP + PPi (IF-1, IF-2, and IF-3)
5. GTP
6. 50S ribosomal subunit
7. Mg2+.
Stage 3: Elongation
of the Peptide Chain Stage 3 : Translocation
Elongation requires 1. The ribosome moves one codon toward the 3’
end of the mRNA
1. the initiation complex This shifts the anticodon of the tRNA attached to the 2nd
codon from the AàP site and the deacylated tRNA from
2. aminoacyl-tRNAs Pà E site

3. a set of three soluble 2. This movement required EF-G (translocase)

and the energy is provided by the hydrolysis
cytosolic proteins called GTPàGDP+Pi
elongation factors (EF-Tu, 3. The uncharged tRNA is dislocated from the E-
EF-Ts, and in bacteria) site and a peptide bond is formed between
4. GTP the growing chain and the new amino acid
carried by the tRNA at the A-site

Stage 4: Termination of Summary of the

Synthesis 5 stages of
1. Elongation continues until the last
amino acid in the sequence
protein synthesis
2. Termination is signaled by the presence
of one of the 3 stop codons UAA, UAG
or UGA immediately following the final
coded amino acid
3. Once the terminal codon occupies A-
site, three termination or release
factors RF1, RF2 and RF3 contribute to
i. Hydrolysis of terminal peptidyl tRNA bond
ii. Release of polypeptide from P-site
iii. Dissociation of the 70S ribosome into the
30S and 50S subunits