N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia

sdvarf@sky.chph.ras.ru

Prebiological synthesis and evolution of macromolecules

Sergey Varfolomeyev

Origin of Life = Origin of self Proliferating Macromolecules

Life phenomena from point of view of polymer chemistry

Polycondensation – the same reaction for all biopolymers

Three-functional monomers Immense decrease of number of

macromolecules comparing to the number of possible variants

Decrease of macromolecules number

Biological systems implemented the extreme low part of possible macromolecular structures

Human genome 3.104 genes

Possible variants of protein structures (20 amino acids, 100 monomers in chain)

20 100 ~ 10 130

Natural (biological) polymers – products of polycondensation of three-functional monomers

Amino acids - polyamides (proteins)

Nucleotides - polyesters (DNA, RNA)

N

NN

N

NH2

O

HO

HH

HH

PO

O-

HO

O-

H2N CH C

CH2

OH

O

CH2

C

OH

O

N

NH2

ON

O

OHO

HH

HH

PO

O-

HO

O-

Copolymerization of three-functional monomers

A

YX

B

X

A

YX

B

YX+

- Interaction of A and B

minimum two types (A, B)

- electrostatic

- hydrophobic

- hydrogen bonds

Y -H2O

All biomacromolecules (products of polycondensation) are unstable in water

Is it possible to have polycondensation process in water?

Thermocycling

Thermocycle – natural global cycle

ΔT - 200 - +200 - 20 - + 150 0 - + 120

Polymerization at high temperatures 120 – 200 OC (statistical number of polymers with different structures

and size) Reduction of temperature, solubilization in water

condensate Macromolecular selection of monomers by polymer

matrix Temperature increasing, polymerization, monomers on

matrix (template), formation of partly complementary chain

Natural (biological) polymers – productsof polycondensation of three-functional monomers

Amino acids - polyamides (proteins)

Nucleotides - polyesters (DNA, RNA)

"Кинетика как наука о законах и механизме развития различных процессов в природе находит в области биологии почти неограниченные перспективы для теоретических исследований и практического применения."

Н.М. Эмануэль, 1966

Copolymerization of three-functional monomers

Kinetic theory describing and explain the dramatic reduction of the number of polymer structures in systems.

S.D.Varfolomeyev,Kinetic models of the prebiological evolution of macromolecules.Thermocycle as motive force of the process,Mendeleev Communications,2007,17,7-9

Copolymerization of three-functional monomers

dNi\dt =kiNiki=kfi-kdi

ki >0 автоселекцияki <0 деградация

Комплексообразование мономера с полимером,как правило,стабилизирует макромолекулу против термической деструкции и гидролитического расщепления

Copolymerization of three-functional monomers

Copolymerization of three-functional monomers

Copolymerization of three-functional monomers

1

2

3

- segregation

- evolution

- autocatalysis, convergence explosion

part of the "positive" molecules

Copolymerization of three-functional monomers

Thermocycle is a method to change absolutelyimprobable events (probability 10 -50) to absolutely certain ones

(probability 1).S.Varfolomeyev,Mendeleev Comm.2007,17,7-9

Adsorption of monomers over polymer Formation of a complex between

monomer and polymer tends to stabilize macromolecule against thermal destruction and hydrolysis

Synthesis of new polymer is selective

Synthesis

BA A+H2O

BA

A

Hydrolysis

Selection of monomers on polymer and synthesis of partly complementary chain

Polymerization

Hydrolysis

Triads of two monomers

Synthesis on polymer

Reactions

Equations

Equations

Increased stability of certain triplets

AAA

BBB

BBB

Accumulation of AAA and BBB, no monomer competition

BBA

AAB

AAA

AAA

BBA

Depletion of AAB due to monomer competition with more stable BBA

Selection principles(competitive advantages)

Greater thermodynamical stability

Greater hydrolysis resistanceVelocity of catalytic processes:

The most stable and «quick» wins!

Experiments

Thermogravimetric analysis (TGA)

Kinetics of weight reduction (water elimination) during the L-asp polycondensation

time, s

wei

gh

t, %

Unique for Russia combined mass-spectrometer ICR + ionic trap Finnigan LTQ-FT

Линейная ионная ловушка

FTMS Data

Магнит7 T

Электронная пушка

ИК-лазер

F = q(E + v x B)

F F

v

Bv

ω = qB/m –циклотронная частота

F = q(E + v x B)

F F

v

Bv

ω = qB/m –циклотронная частота

FT_time_domane2_FFT_FT #1 RT: 65.39 AV: 1 NL: 2.13E5T: FTMS + p ESI Full ms [ 110.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

648.85

432.90

338.34

679.811296.67

998.96

784.41508.26

619.36

908.75324.43 1859.03

716.80

216.45 1028.53 1358.601237.09 1632.901453.75

World of polypeptidesneg-F8p_10e3_200-2000_FT #1 RT: 18.02 AV: 1 NL: 3.84E3T: FTMS - p ESI Full m s [ 200.00-2000.00]

300 400 500 600 700 800 900 1000 1100 1200 1300

m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

592.138z=0

477.112z=0

707.165z=1

822.192z=1

574.128z=2

513.861z=0

937.218z=1358.841

z=0689.154

z=2459.101z=2

804.182z=2 959.200

z=1919.207

z=0380.823

z=0729.147

z=0

844.174z=0

614.120z=0

1052.245z=1

1074.226z=0755.674

z=0866.156

z=0 981.181z=0306.879

z=0 1167.273z=0

1096.212z=0

1233.624z=0

MS/MS approach

Sequencing of products of polycondensation

H2N CH C

(CH2)4

O

NH2

HN CH C

(CH2)4

OH

O

NH2

HN CH C

(CH2)4

N

O

NH2O

O

N

O

O

CCHNH2

(CH2)4

O

NH2 n

n = 7, 8

H2N CH C

(CH2)4

N

O

NH2O

O

NH

4O

O

HO

N

O

O

N

O

O

4

H2N CH C

(CH2)4

N

O

NH2O

O

4

NH

CH C

(CH2)4

O

NH2

HN CH C

CH2

N

OO

OO OH

H2N CH C

(CH2)4

N

O

NH2O

O

5

NH

N

O

O

3

N

O

OO

HO

O

H2N CH C

(CH2)4

N

O

NH2O

O

5

NH

N

O

O

NH

HC COOH

CH2

HOOC

O

O

HO

H2N CH C

(CH2)4

N

O

NH2O

O

NH

O

N

O

O

N

O

O

2

6

O

HO

H2N CH C

(CH2)4

N

O

NH2O

O

N

O

O

8

Sample G1

H2N CH C

(CH2)4

OH

O

NH2

L-Lys

H2N CH C

CH2

OH

O

C

OH

O

+

L-Asp

104 hours, H2SO4

1920 possible variants of structures, 9 possible structures were realized.

H2N CH C

(CH2)4

OH

O

NH2

H2N CH C

(CH2)4

O

NH2

HN CH C

(CH2)4

OH

O

NH2

L-Lys

Sample G1

HN CH C

(CH2)4

N

O

NH2O

O

N

O

O

CCHNH2

(CH2)4

O

NH2 n

n = 9

H2N CH C

(CH2)4

N

O

NH2O

O

N

O

O

13

H2N CH C

CH2

OH

O

C

OH

O

+

L-Asp

HN CH C

(CH2)4

N

O

NH2O

O

N

O

O

CCHNH2

(CH2)4

O

NH2 n

n = 8

19456 Possible variants of structures , 4 possible structures were realized.

278 hours, H2SO4

World of polypeptides

World of polypeptides

Sample G3

H2N CH C

(CH2)4

OH

O

NH2

L-Lys

H2N CH C

CH2

OH

O

C

OH

O

+

L-Asp

860 Possible variants of structures , 11 possible structures were realized.

104 hours, HCl H2N CH C

(CH2)4

N

O

NH2O

O

N

O

O

3H2N CH C

(CH2)4

NH

O

NH2

NHO

O

HO

N

O

O

N

O

O

6O

O

HO

H2N CH C

(CH2)4

NH

O

NH2

N

O

O

N

O

O

6

O

O

HO

H2N CH C

(CH2)4

N

O

NH2O

O

NHO

O

HO

NH

O

O

N

O

OHO

NH

OHO

O OH

H2N CH C

(CH2)4

N

O

NH2O

O

NHO

O

HO

NHHC

O

OH(CH2)4

NH2

NH2-Lys-Asp-Asp-Lys-Asp-Asp-Asp-Lys-Asp-CO2H NH2-Lys-Lys-Asp-Asp-Asp-Asp-Asp-Lys-Asp-CO2H

NH2-Lys-Asp-CO2H

NH2-Lys-Asp-Asp-CO2H

NH2-Lys-Lys-Asp-CO2H

NH2-Lys-Lys-Asp-Asp-CO2H

Sample G3

H2N CH C

(CH2)4

OH

O

NH2

L-Lys

H2N CH C

CH2

OH

O

C

OH

O

+

L-Asp

128 Possible variants of structures , 4 possible structures were realized.

278 hours, HCl

H2N CH C

(CH2)4

N

O

NH2O

O

N

O

O

6

H2N CH C

(CH2)4

NH

O

NH2

N

O

O

N

O

O

5

O

O

HO

H2N CH C

(CH2)4

N

O

NH2O

O

NH

O

O

2

HO

N

O

O

2

NH

HC COOH

(CH2)4 NH2

NH2-Lys-Asp-COOH

Lys-Asp, basic solution

Chain 5 monomers: 9 structures of 60 possible

Chain 7 monomers: 11 structures of 252 possible

World of polypeptides

KK DK

DDK KDK KKK

DKDK KDKK DKDD

DKDDK DKKDK KDKKK

All structures are closely related

Complimentary structures or intermediates

NH2-AspAspLys-COOH NH2-Asp-Asp-Lys-Asp-Asp-Asp-COOH

NH2-Asp-Asp-COOH NH2-Asp-Asp-Asp-Asp-COOH

NH2-Lys-Lys-COOH NH2-Lys-Lys-Lys-COOH

General principles of evolution of multifunctional macromolecules

Non-statistical distribution of monomers along the polymeric chain

The primary synthesized polymers works as a template for sorption and subsequent polymerization

Selection factors (stability, catalysis, etc.)

A

X Y

B

X Y,

Termocycling

Evolution to unique structure, dramatic reduction of the number

of structural variants.

Globular peptides and proteins

Flexible polyanion RNA

Rigid-linear polyanion DNA

Interdependent coexistence of three worlds of pre-biopolymers

++

++

-

-

-

- -

-

-

-

-

-

-

-

-

In the regime of hydrolytic degradation, system accumulates peptides and nucleic acids affine to each other and forming stable supramolecule complexes

Formation of stable suprmolecule complexes is a selective factor

k1 k2k3

Degradation

k1,k2> k3

Interdependent coexistence of three worlds of pre-biopolymers

Accumulation of selected limited repertoire of peptides (pre-proteins)

Accumulation of selected set of complexes of peptides with RNA

Transfer of structural information from RNA to peptide (and vice versa) and fixation in form of double-stranded DNA, molecular hieroglyphs

Polymer-RNAcomplex

RNA DNA DNA

Interdependent coexistence of three worlds of pre-biopolymers

The same pattern for three main groups of biopolymers

Thermocycle (phase transfer (- H2O)) is a driving energetic force of the process and provides thermodynamics possibility of polycondensation

Autoselection of polymers composed of three-functional monomers. Polymer always makes influence on the products composition due to supramolecular interactions of monomers and polymers

This drives switch from statistical to evolutionary-driven synthesis

Proteins and peptides

RNA

DNA

General principles of evolution of multifunctional macromolecules

(1) Non-statistical distribution of monomers along the polymeric chain

(2) The primary synthesized polymers work as the template for sorption and subsequent polymerization

(3) Selection (stability, catalysis, etc.)

A

X Y

B

X Y,

Termocycling

(4) Evolution to unic structure, dramatic reduction of the number

of structural variants.

Трифункциональные мономеры (X Y, X Y)

Предбиологическая эволюция биополимеров

Поликонденсация - базовый химический процесс (- H2O), образование амидов, пептидов, белков, фосфодиэфирной (РНК, ДНК) связи

Термоцикл (фазовый переход (- H2O)) – движущая энергитическая сила процесса – обеспечение термодинамической возможности поликонденсации

Предбиологическая эволюция биополимеров

Автоселекция полимеров из трифункциональных мономеров

Полимер ВСЕГДА оказывает влияние на состав продуктов в силу супрамолекулярного взаимодействия мономера с полимером

Переход от чисто статистического к эволюционно-направленному синтезу

Общие закономерности для всех трех основных классов биополимеров

- Пептиды и белки О , рибонуклеиновые кислоты (), дезоксирибонуклеиновые кислоты ( I I )

Большая термодинамическая устойчивость Большая устойчивость к гидролитической деструкции Каталитические свойства

Гидролиз пептидов Гидролиз полинуклеотидов Гидролиз пирофосфата Получение мономеров Поликонденсация аминокислот Поликонденсация нуклеотидов

Предбиологическая эволюция биополимеров

Принципы отбора(конкурентные преимущества)

Побеждает наиболее стабильный и «быстрый»

Подвижно – линейные - РНК

Жестко - линейные - ДНК

Взаимозависимое сосуществование трех «миров» предбиополимеров

Глобулярные – пептиды, белки

+ +

++-

--

-

--

-

--

--

-

-

Взаимозависимое сосуществование трех «миров» предбиополимеров

В режиме гидролитической

деградации в системе

накапливаются пептиды и

нуклеиновые кислоты, которые

афинны друг к другу, то есть

образуют устойчивые

супрамолекулярные комплексыОбразование стабильных супрамолекулярных комплексов как фактор отбора

+

k1

k2 k3

деградация

k1,k2> k3

Накапливается «отселектированный» ограниченный набор структур пептидов (предбелков)

Накапливается «отселектированный» набор комплексов пептидов – РНК

Перенос структурной информация от пептида к РНК, и «фиксация» этой информации в ДНК - форме

Взаимозависимое сосуществование трех «миров» предбиополимеров

+

Комплекс Полимер - РНК

РНК Цепь ДНК

ДвойнаяСпираль ДНК

Молекулярные иероглифы

Ферменты и активные центры ферментов Антитела Рецепторы Малые РНК

The great paradox of enzymes origin

Very restricted number of catalytical sites structures

2 Metal ions (2 Zn)\HDB Parathion hydrolase (1DPM)

Chymotrypsin and Streptogrisin

: Primary structures are absolutely different

Catalytical sites are the same

Organophosphatehydrolase

(EC 3..1.8.1)X

R2

P

R2

ZR3R1 O P OHR1 O + HZR3

H2O

Organophosphate hydrolase

O

X = O, S Z = O, S and F, when R3 is absent

Reaction catalyzed by

organophosphate hydrolase (EC 3.1.8.1)

An important conclusion: The role of Asp from catalytic triad is to orient His, but not to serve as a proton relay

2.69Å

2.62Å

2.62Å

2.72Å

Asp102

Ser214

Ala55

Ser195

His57

X-ray structure 2PTC

Переход от гидролитических реакций к синтетическим-

Одни и те же каталитические структуры осуществляют гидролиз и синтез

Structural Unity of catalytical sites

Pyrophosphatase Exonuclease DNA- polymerase

N.M. Emanuel Institute of Biochemical Physics, RAS, Moscow, Russia

sdvarf@sky.chph.ras.ru

Prebiological synthesis and evolution of macromolecules

S.D. Varfolomeev