Transcript S - IEA

MAY 2006
Pier Luigi Luisi
UniRoma3
THE ORIGIN OF LIFE ON EARTH:
WHAT SCIENCE HAS TO SAY
the “continuity
principle”
no unbridgeable gap between inorganic and living matter;
each stage in evolution develops continuously from the
previous one, at each stage there is a continuous path
backwards to the prebiotic state and forward to modern
organisms
Orgel; Morowitz; de Duve
The traditional definition:
Science is
the enterprise
to explain the phenomenology
of the world in terms of the laws
of physics and chemistry
..not the only way to explain the world
The science of the origin of life has to
adopt the deterministic, continuity viewotherwise it would not be possible to
adopt a scientific method of inquiry
Christian de Duve
Harold Morowitz
And others…
WHAT CAN SCIENCE SAY ABOUT
THE BASIC QUESTION:
A maze illustrating the chemical reactions that interconvert small molecules in cells.
NUTRIENTS / ENERGY
N
C
wall
components
N
B
A
C
C
C
C
M
H
DNA
H
G
D
RNA
Q
Proteins
C C
C
C
various organells
THE CELL‘S MAIN ACTIVITY IS
TO MAINTAIN ITS OWN IDENTITY
(SELF-MAINTENANCE)
DESPITE THE ENORMOUS NUMBER
OF TRANSFORMATIONS
AND THE CELL DOES SO
THANKS TO A PROCESS OF
RE-GENERATION FROM WITHIN
The cellular definition of life:
A system spatially defined by a
boundary of ist own making
and that is self-sustaining
by re-generating all system‘s components
from the inside
A DISCOVERY: THE CELLULAR
(AUTOPOIETIC)
DEFINITION OF LIFE
ALSO APPLIES
TO MULTICELLULAR,
MACROSCOPIC
LIFE
THE GAME OF THE TWO LISTS
LIST OF THE LIVING
LIST OF THE NON-LIVING
The fly
The radio
The tree
The automobile
The mule
The robot
The baby
The crystal
The mushroom
The moon
The amoeba
The computer
QUESTION:
What discriminates the living from the non-living?
IN OTHER WORDS: What is the quality (or qualities) which is present in all members
of the "living list" and which is not - and cannot - be present in
any of the elements of the "non-living" list?
(movement? growth? reaction to stimuli? reproductivity? processing of energy arriving from outside? etc....)
SOME MAIN ASSUMPTIONS OF PRESENT
DAY RESEARCH ON THE ORIGIN OF LIFE
1. Life originated from inanimate matter as a spontaneous and continous increase of molecular complexity. LIFE
Chemical continuity principle - no transcendental
principle.
2. The chemical process(es) to transition to life can be
reproduced in the laboratory with the presently
available chemical techniques and chemicals.
inanimate
3. And this can be implemented in a reasonable
matter
(hours or max. days) experimental time span once you know the right combination of prebiotic compounds
and theconditions.
4. Since there is no documentation on how things really
happened, there is no obligatory research pathway.
Aleksandr Oparin ( 1894 – 1980 )
Water
vapor
Electrodes
discharge sparks
("lightning
simulation")
CH4
H2O
NH3
H2
Molecule
Formic acid
Glycine
Alanine
Lactic acid
Acetic acid
Condenser
Stopcocks
for testing
of samples
Sarcosine
Out
Succinic acid
Urea
Cold water
inlet
Heated water
("ocean")
Condensed liquid
with complex
molecules
Glutamic acid
Aspartic acid
1
10
100 1000
Yield in micromoles
Synthesis of purines
Oró, 1960
CN
H2N
H2N
NC
H2N
CN
diaminomaleonitrile
N
adenine
H2N
HCN
N
H
N
N
H
diaminopurin
O
N
HN
N
NCO
(CN)2
O
N
N
H2N
N
H2N
N
H
NH2
N
H
H2O
(CN)2
NH2
N
N
4-amino-imidazole-5-carbonitrile
HCN
N
O
N
H
hypoxantine
H2N
O
N
HN
N
guanine
-
N
H
N
HN
H2N
NH
N
H
xanthine
cells
metabolic networks
polymer complexes
macromolecules
biomonomers
molecules
atoms
About the philosophical
framework
IS THE ORIGIN OF LIFE ON EARTH AN
OBLIGATORY
PATHWAY (IT HAD TO HAPPEN!)
OR
IS IT DUE TO CHANCE?
Determinism vs contigency
in the origin of life
…”I favor the view that life was bound to arise
under the physical-chemical conditions that surrounded its birth”
De Duve, 2002
We have no reason to believe that biogenesis
was not a series of chemical events subject
to all of the laws governing atoms and their interactions.”
Morowizt, 1991
“..It is self-evident that the universe was pregnant
with life and the biosphere with man.
Otherwise, we would not be here.
Or else, our presence
can be explained only by a miracle…”
De Duve, 2002
Other „crypto-creationists“????
Anthropic principle
Panspermia
SETI
THE ANTHROPIC PRINCIPLE....
“If
life follows from (primordial) soup with
causal dependability, the laws of nature
encode a hidden subtext, a cosmic
imperative, which tell them:
‘Make life! And, through life, its by-products,
mind, knowing, understanding…’”.
Paul Davis, 1991
OPPOSITE TO
THAT....
CONTINGENCY
origin
of earth
origin
of life
4.5 billion
years
{
aer.photosynt.
2.5
O2 in atmosph.
{
eucaryotes
sexual
reproduction
{
{
first fish
first mammals
primates
Neanderthal
3.5
1.5
}
...run the tape again,
and the first step from
procaryotic to eucaryotic
cell may take 12 billion
years instead of 2...
Stephen J.Gould
in "Wonderful Life"
1991 Penguin Science
unicellular
organisms
PRECAMBRIAN
0.5
}
now
multicellular
organisms
WE WOULD LIKE TO THINK OURSELVES
NECESSARY, INEVITABLE, ORDAINED FOR
ALL ETERNITY.
ALL RELIGIONS, ALL PHILOSOPHIES, AND
EVEN PART OF SCIENCE TESTIFY
TO THE UNWEARYING, HEROIC EFFORT
OF MANKIND
DESPERATELY DENYING
ITS OWN CONTINGENCY
J.Monod, Chance and Necessity, 1971
ARE WE ALONE IN
THE UNIVERSE??
GENERALLY, CONTINGENCY
AND DETERMINISM PROCEED THE HAND IN
THE HAND
...THERE CANNOT BE CONTINGENCY WITHOUT
RESPECT FOR THE DETERMINISTIC LAWS OF NATURE
THE IMAGE OF THE RIVER FLOWING DOWNHILL...
HOWEVER, THE BASIC QUESTION OF THE RELATIVE
IMPORTANCE REMAINS
CAN YOU DO EXPERIMENTAL RESEARCH
ON THESE ITEMS OF
DETERMINISM
VS CONTINGENCY?
...CONSIDER THE
PROTEINS OF
LIFE
SCHEMATIC REPRESENTATION OF THE
THREE-DIMENSIONAL STRUCTURE OF I gG
antigenbinding
site
Fab-unit
Fc-unit
Fab-unit
antigenbinding
site
carbohydrate
residue
Each amino acid residue is represented by a small circle. The H chains are red
and the L chains blue. A carbohydrate residue is yellow.
E. W. Silverton, et al. Proc. Nat. Acad. Sci. 74
(1977); p. 142.
CO
NH
C H CO
NH
CH
R1
X
CO
NH
CH
R2
Y
Z
R3
P
Y
O
P
O
O
Rib
Base1
O
P
O
Rib
Base2
O
P
O
Rib
Base3
On the importance of being a copolymer
(
CO
CH
)n
NH
Ri
Calculate:
How many different macromolecules
can you build, when
n = 60 and i = 1 - 20
60
20 x 20 x 20 x .....
evolution
N = 2060
~
1070
!!!
energetics
In nature there are only 1012 - 1014 proteins
space of the proteins
present in nature 10 10
(radius ca. 1 atom)
radius of the
universe
space of all
possible proteins
10130
(20100)
Cast the dice again and........
And you will get a different set
of macromolecules
that do not necessarily support life
QUESTION:
ARE THE PROTEINS OF LIFE THE ONLY
ONES THAT COULD BE FORMEDAND GAVE ORIGIN
TO LIFE BY A DETERMINISTIC
(OBLIGATORY)
SERIES OF EVENTS
OR
ARE THEY THE PRODUCT OF
CONTINGENCY(CHANCE)
AND LIFE IS ALSO A PRODUCT OF
CONTINGENCY?
PROJECT RANDOM POLYPEPTIDES:
MAKE A VASTE LIBRARY OF RANDOM
DE NOVO PROTEINS
ASK THE QUESTION: HOW MANY OF
THEM WILL BE
FOLDED?
Folding is the prerequisiste for any
functionality of proteins
PHAGEMID VECTOR SYSTEM
IN PHAGE DISPLAY
Helper Phage
Modified Phage
gene 3
fusion protein
Plasmidic
DNA
ss DNA
gene 3
fusion
ANALYSIS OF THE PEPTIDE
PHAGE LIBRARY
Library
1.98 x 109 clones
180 clones were randomly selected
44% correct insert (79 clones)
73 clones tested for thrombin stability
% of the73 clones of the phage library
DISTRIBUTION OF THE PEPTIDE
LIBRARY WITH RESPECT TO
THROMBIN DIGESTION
30.00
“Stable”
25.00
“Unstable”
20.00
15.00
10.00
5.00
0.00
0-10
10-20
20-30
30-40
40-50
50-60
60-70
% digestion categories
70-80
80-90
90-100
TRIDIMENTIONAL STRUCTURE PREDICTION
Cys residues
C-terminal
FLUORESCENCE STUDIES
Trp 23
Fluorescence (standardized)
C-terminal
Wavelength (nm)
• The Trp residue could be exposed to the solvent
N-terminal
Temptative conclusion suggested by these data:
„Our“ proteins have nothing special from the
structural or
thermodynamic point of view
One may therefore suggest that they are
the products of contingency
SOME MAIN ASSUMPTIONS OF PRESENT
DAY RESEARCH ON THE ORIGIN OF LIFE
1. Life originated from inanimate matter as a spontaneous and continous increase of molecular complexity. LIFE
Chemical continuity principle - no transcendental
principle.
2. The chemical process(es) to transition to life can be
reproduced in the laboratory with the presently
available chemical techniques and chemicals.
inanimate
3. And this can be implemented in a reasonable
matter
(hours or max. days) experimental time span once you know the right combination of prebiotic compounds
and theconditions.
4. Since there is no documentation on how things really
happened, there is no obligatory research pathway.
The problem of re-making
our present macromolecular sequences
Their synthesis and the reconstitution of life by the
bottom-up approach is made difficult or impossible
by the laws of contingency :
..you can make different ones, and show that in
principle this pathway is possible
THE ALTERNATIVE APPROACH TO
THE CONSTRUCTION OF THE
MINIMAL CELL:
USE THE EXTANT NUCLEIC ACIDS
AND PROTEINS
..this is not the approach to the origin of
life
the notion of the minimal cell:
lip idic b ilay er
cont aining t he minimal and suf f icient
number of component s t o be " alive"
self -m aint enanc e
alive
reproduct ion
mut at ion
The work on the minimal living cell
Is connected with the study of the
early cells - namely the protocells at
the time before the advent of ribosomes
and before the high selectivity
of modern times —
First step:
find a suitable compartment as cell
model
And show that this is capable of hosting the
Complex molecular biology reactions of life
Liposomes, as closed spherical
bilayers, are considered the most
likely precursors of early living cells
(protocells)
LIPOSOMES ARE JUST TINY SOAP BUBBLES
Cross-sectional views of the three structures that can be formed by
mechanically dispersing a suspension of phospholipids in aqueous solution
Micelle
Liposome
Bilayer sheet
The red circles depict the hydrophilic heads of phospholipids, and the
squiggly lines (in the yellow region) the hydrophobic tails.
Nature 357(1992)57
H2 O
S
S
S
S
s-s excess
H2O
S
water-insoluble
surfactant precursor
bound to the bilayer
S
S
S
S
S
hydrolyzed
on / by
the bilayer
lipophilic bilayer
of surfactant S
s-s
S
S
S
S
larger
unstable
intermediates
S
S
S
S
S S
S
S
S
S
S
S
S
S
H 2O
S
forming more
of smaller stable vesicles
S
S
H 2O
S
S
S
S
S
S
S
S
H 2O
S
S
S
S
S
S
S
S
NOW, LET US PUT SOMETHING
IMPORTANT
INSIDE...
Is this life?
Not really.
With progressive
generations, the active components
are diluted out
because they are not fabricated
by the compartment itself
..death
by dilution....
Step two:
Protein synthesis inside liposomes
PROTEIN EXPRESSSION IN LIPOSOMES (MOSTLY
GREEN FLUORESCENCE PROTEIN) HAS BEEN
DESCRIBED BY SEVERAL GROUPS:
Oberholzer et al., 1999, 2001
Yomo et al., 2001
Tsumoto et al., 2001
Fischer et al, 2002
Nomura et al., 2003,
Pietrini et al., 2004
Noireaux et al, 2004
Ishikawa et al, 2004
a n e x a m p l e of m i n im a l c e l l :
a ce l l w hi c h cr e a t e s it s b ou n da r y
S
S
A
S
S
S
A
E
S
S
S
S
S
S
S
Protein expression inside the liposomes
Working plan
1.
Expression of green fluorescent protein (GFP)
or any other simple model protein
GFP
plasmid
2.
A
Expression of the enzymes
that make the liposome
A
L
L
L
A
E
L
L
L
L
L
L
L
L
L
L
L
L
L
L
lipids
L
L L
L LL
L
L L L
L L
L
L
L
L
L L
L L
L
L
L
L
L
L
L
L
L
(dead)
Protein expression inside the liposomes
and next step (minimal cell)
Working plan
3.
Production of the boundary as in 2., and with self-reproduction
of the ribosomes and of the DNA-machinery
S
S
S
A
S
S
S
S
S
S
S
S
S
E
A
S
S
S
S
S
S
S
S S
S S
S
S
S
S S
SS
S
S
S
S
S
S
S
S
S
S
S
How many genes are then necessary?
S
Presently: work with THE PURE SYSTEM,
A kit with only 37 enzymes
(plus the ribosomes)
capable of expressing proteins
Developed by Ueda and coworkers in Tokyo
in th early 2000
Nature 409, 387 - 390 (2001)
Synthesizing life
JACK W. SZOSTAK, DAVID P. BARTEL & P. LUIGI LUISI
THE NEW RESEARCH AREA ON THE MINIMAL
CELLS INTERESTS NOW ABOUT ONE DOZEN
GROUPS AROUND THE WORLD.
WHY IS THIS RESEARCH RELEVANT?
1. UNDERSTANDING THE CHEMICAL ESSENCE
OF LIFE BY RECONSTRUCTING IT IN THE LAB
2. UNDERSTANDING OF THE EARLY CELLS
3.BIOTECHNOLOGICAL RELEVANCE (E.G.,
PROTEINS SYNTHESIS WITH SIMPLE LIPOSOME
SYSTEMS
AS A WAY OF CONCLUSION
CONCERNING THE TRANSITION TO LIFE FROM THE
INANIMATE MATTER:
1. IT HAS NOT BEEN IMPLEMENTED IN THE LAB YET.
THEREFORE, IT REMAINS AN HYPOTHESIS.
AND THE BOTTOM UP APPROACH SEEMS TO BE MADE
IMPOSSIBLE BY THE LAWS OF CONTINGENCYCONCEPTUALLY AND EXPERIMENTALLY
2.THE CONSTRUCTION OF SYNTHETIC LIVING CELLS
APPEARS POSSIBLE USING EXTANT MACROMOLECULES.
MOST SCIENTISTS BELIEVE, THAT „SOON“,
THIS WILL BE REALIZED.