THE WORLD OF THE“NEVER BORN PROTEINS”

by Cristiano Chiarabelli

Lightnings_SV3.aviLightnings_SV3.avi

Electrodesdischarge sparks("lightningsimulation")

Watervapor

Stopcocksfor testingof samples

Heated water("ocean")

Condensed liquidwith complexmolecules

Cold waterinlet

Out

Condenser

CH4H2ONH3H2

Glutamic acidAspartic acid

Urea

Succinic acid

Sarcosine

Acetic acid

Lactic acidAlanine

GlycineFormic acid

Molecule

1 10 100 1000Yield in micromoles

radius of the universe

space of allpossible proteins 10130

(20100)

space of the proteins present in nature 1010 (radius ca. 1 atom)

There may be an entire universe of “Never Born Proteins” (NBP), whose properties have never been sampled by Nature

Contingency theory:

extant proteins are the result of the simultaneous interplay of several concomitant causes (Gould, 1994).

Determinist theory:

The life constituents are the result of an evolutive fine work; what we see is the better possible solution for the biological needs (de Duve, 1995).

The proteins existing on our Earth are only an infinitesimal fraction of the theoretically possible ones (Xia & Levitt, 2004).

Natural proteinsPossible-protein space

THEORETICAL CONTEXT

SOME BASIC CALCULATIONS WITH A 50 AMINO ACID PEPTIDE . . . .

1.1 X 101.1 X 1065 65 POSSIBLE AMINO ACID COMBINATIONSPOSSIBLE AMINO ACID COMBINATIONS SYNTHESIS OF ONE MOLECULE OF EACH COMPOUND: SYNTHESIS OF ONE MOLECULE OF EACH COMPOUND:

– 1.1 X 101.1 X 104242 KG OF MATERIAL KG OF MATERIAL

– 1.8 X 101.8 X 101717 TIMES THE WEIGHT OF THE EARTH TIMES THE WEIGHT OF THE EARTH

IT SEEMS UNLIKELY THAT NATURE TRIED ALL TRIED ALL COMBINATIONSCOMBINATIONS

IT IS POSSIBLE TO SAMPLE AGAIN AND ISOLATE IT IS POSSIBLE TO SAMPLE AGAIN AND ISOLATE FOLDED PEPTIDES WITH NO HOMOLOGY TO RECENT FOLDED PEPTIDES WITH NO HOMOLOGY TO RECENT PROTEINSPROTEINS

Selection

AIM OF THE PROJECT

FOLDING FREQUENCY DETERMINATION OF RANDOM FOLDING FREQUENCY DETERMINATION OF RANDOM

50aa POLYPEPTIDES PRESENTING NO HOMOLOGIES 50aa POLYPEPTIDES PRESENTING NO HOMOLOGIES

WITH EXTANT PROTEINSWITH EXTANT PROTEINS

STRUCTURAL PROPERTIES CHARACTERIZATIONSTRUCTURAL PROPERTIES CHARACTERIZATION

STRATEGY FOR THE ISOLATION OF FOLDED PEPTIDES: PHAGE DISPLAY

Random Peptide (50mer)

c-mycTAG

mAB9E10

Thrombin cleavage site

pIII

P R G

ssDNA

pVIII

Schematic drawing of the filamentous Phage M13

•The filamentous phage M13 is used in phage display•In A phage the DNA information is “linked“ to the protein•The single-stranded DNA genome is packaged in a long cylinder•M13 is able to infect E. coli•Display of foreign peptides or proteins on pIII•Used in affinity maturation of peptide and protein ligands•Sensitive assay: selection of single phage particles

PHAGE DISPLAY TECHNIQUE [1]

C-myc Gene III

Ori M

13

Ampr

Phagemid vector

Col

E1

Random fusion protein gene

Fusion random gene

Phagemid

Random fusion protein

p III

Phagemid DNA

g III

[1] Smith, G.P. Science. 1985

g IIIss DNA

Helper Phage (M13K07)

II X

V

VII

IX

I VI III VIII

IV M13K07

p III

VALIDATION OF OUR TEST SYSTEM WAS DONE WITH APP

APP (Avian Pancreas Peptide - Hormone) protein

• 39 aa• NO DISULFIDE BRIDGES• FOLDED• STRUCTURE KNOWN

STRUCTURE OF AVIAN PANCREAS PEPTIDE (APP)

Turn

Proline helics (PXXP)

-helics

DESIGN OF THE PHAGEMIDDESIGN OF THE PHAGEMID

Ori M13

Ap

ColEI

gene III

APPlinker

Export signal

VPRG PRG RG No

c-myc tag

THE RESTRICTION SITES VPRG, PRG, RG

TAG

mABShort flexible linker VPRG

PRG RG

APP

THROMBIN ASSAYTHROMBIN ASSAY

Phages were incubated 18 hours withPhages were incubated 18 hours with

– 0.0 U/ml Thrombin0.0 U/ml Thrombin– 0.15 U/ml Thrombin0.15 U/ml Thrombin– 1.5 U/ml Thrombin1.5 U/ml Thrombin

Binding assay enrichment (ELISA)Binding assay enrichment (ELISA)

Samples were used to infect E. coliSamples were used to infect E. coli

Colonies were counted and frequencies were calculatedColonies were counted and frequencies were calculated

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

No PR PRG VPRG

DIFFERENT THROMBIN CLEAVAGE SITES INSERTED UPSTREAM OF APP

0.0 U/ml 0.15 U/ml 1.5 U/ml

% d

iges

tion

by

Thr

ombi

n

CONCLUSIONS

•Phage with no cleavage site for Thrombin is not affected by

incubation with the enzyme

•The sequence RG is not a good Thrombin Substrate

• VPRG and PRG are equally good Thrombin Substrate

THE PROTEASE CLEAVAGE SITE OF CHOICE IS: PRG

NEXT IMPORTANT STEP . . .NEXT IMPORTANT STEP . . .

proof that Thrombin does not cleave the PRG proof that Thrombin does not cleave the PRG sequence in a folded peptidesequence in a folded peptide

THE RESTRICTION SITE PRGPSQ

TAG

mABshort linker

P R G

APP

Turn

P R G

THE RESTRICTION SITE PRGPTY

TAG

mABshort linker

APP

0.0 U/ml 0.15 U/ml 1.5 U/ml

% d

iges

tion

by

Thr

ombi

nPRG INSERTED AT DIFFERENT POSITIONS IN APP

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

PRG PRGPSQ PRGPTY

CONCLUSIONS

- THE MUTANT PROTEINS PRGPTY AND PRGPSQ ARE BETTER

PROTECTED AGAINST THROMBIN CLEAVAGE THAN PRG

- THE PROLINE HELICS OF APP IS NOT DESTROYED BY THE

INSERTION OF PRG AT THE TWO POSITIONS

A RANDOM FOLDED PROTEIN WITH THE SEQUENCE PRG INSIDE CAN BE SELECTED USING

THROMBIN

RANDOM LIBRARY CONSTRUCTIONRANDOM LIBRARY CONSTRUCTION

Pro - Arg - Gly

digestion and ligation

C-myc

Gene III

NotINcoI

XhoI XbaIBamHI

C-myc

XbaINotI XhoI XhoINcoI BamHI

Gene III

Ori M

13

Ampr

Phagemid Vector Phagemid Vector [1][1]

Col

E1

C-myc Gene III

Random selection of 180 colonies

Sequencing

79 correct plasmids

[1]Lang, S. et al. Biochemistry. 2000

XhoINotI XbaIDNA RANDOM

LIBRARY

XhoI

47 aa random

RANDOM SCHEMA NNK

N ( A, C, G, T) K (G, T)

(4 × 4 × 2)47 = 3247 ~ 1070 theoretical random sequences

Amber mutation: TAG QUAG UGA UAA

Forward OligonucleotidesForward Oligonucleotides

XhoINotI

69 nt XhoI XbaI72 nt

Revers Revers OligonucleotidesOligonucleotides

Electroporation(Electro Ten-blu, amber mutation)

1st position 2nd position 3th position

Exp Theor Exp Theor Exp Theor

T 24.8 25.0 25.1 25.0 51.5 50.0

C 23.0 25.0 24.1 25.0 0.1 0.0

A 26.9 25.0 25.2 25.0 0.1 0.0

G 25.2 25.0 25.6 25.0 48.3 50.0

RANDOMNESS OF PHAGE PEPTIDE LIBRARY AT THE NUCLEIC ACID LEVEL

RANDOMNESS OF PEPTIDE PHAGE LIBRARY AT THE AMINO ACID LEVEL

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr

Theoretical Experimental

% th

at a

n A

A o

ccur

s in

the

phag

e lib

rary

Folded peptides are better resistant to hydrolysis then random-coil onesFolded peptides are better resistant to hydrolysis then random-coil ones

pIII

Random polypeptide

(47 aa)

PRGPRG c-myc THROMBINTHROMBIN

pIII

Random polypeptide

(47 aa)

PRGPRG

c-myc

c-myc

c-myc

Anti-c-myc 9E10

PROTEIN FOLDING SELECTION BY THROMBIN

DISTRIBUTION OF THE PEPTIDE LIBRARY WITH RESPECT TO THROMBIN DIGESTION

0.00

5.00

10.00

15.00

20.00

25.00

30.00

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100

“Stable” “Unstable”

% o

f the

73 c

lone

s of

the

phag

e lib

rary

% digestion categories

POLYPEPTIDE 127 PRIMARY SEQUENCE

Val

Glu Ala AlaAla Glu Gln Leu Ile Ser Glu Asp Leu Asn GlyLys Ala Arg

Cys

CysLeu

Gly

ArgAsn Arg Trp Met Arg MetGlyAsp Gly LeuValTrp Cys Trp ThrHisIleAlaPro Asp

GluAspThr Gln His ThrThrLeu His His GlnLys ArgIle GlnThrSerCys Ala

Cys

Glu

LeuMet

H2N

C

O

OH

c-myc

thro

mbin

Non polar aa – 39%

Charged aa – 21%

Polar aa – 40%

FLUORESCENCE STUDIES

Trp λem = 351

127 λem = 343

127 λem (urea 6M) = 343

One or more Tryptophan residues are localized in a zone less exposed to the solvent

Flu

ores

cenc

e (s

tand

ardi

zed)

Wavelength (nm)

native

Flu

ores

cenc

e (s

tand

ardi

zed)

Wavelength (nm)

native+ urea (6M)

CD STUDIES

CD specta of proteins NBP127. Continue lines for native proteins; dashed lines for proteins at 60 °C; empty triangles for proteins in 6 M urea; empty circles for proteins refolded after 12h dialysis.

C

D

Non polar aa – 55%

Charged aa – 13%

Polar aa – 32%

Cys

Glu Ala AlaAla Glu Gln Leu Ile Ser Glu Asp Leu Asn GlyLys Ala Met

Ala

ArgVal

Gly

ArgCys Glu Gly Ala Ser GlyTrpMet Gly TyrSerPro Pro Gly GlnGlyLeuValPro Ile

ProArgThr Asn Ser ThrThrPhe Ala Pro MetAsp ProGln ValHisLeuArg Ser

Arg

Cys

LeuGln

H2N

C

O

OH

c-myc

thro

mbin

POLYPEPTIDE 1 PRIMARY SEQUENCE

CD STUDIES

CD specta of proteins NBP1. Continue lines for native proteins; dashed lines for proteins at 60 °C; empty triangles for proteins in 6 M urea; empty circles for proteins refolded after 12h dialysis.

TRIDIMENTIONAL STRUCTURE PREDICTION

Globular Structure:Globular Structure:

3 α-Helices:

1. Res. 9 → 22

2. Res. 27 →30

3. Res. 42 → 52 (PRG)

Tot. α-Helix ~ 40%

: no polar zones

: polar zones

: + residues

: - residues

C-terminal

TRIDIMENTIONAL STRUCTURE PREDICTIONCys residues

N-terminal

C-terminalTrp 23

• The Trp residue could be exposed to the solvent

FLUORESCENCE STUDIESF

luor

esce

nce

(sta

ndar

dize

d)

Wavelength (nm)

HIGHLIGHTS

A PHAGE DISPLAY METHOD FOR THE ISOLATION OF A PHAGE DISPLAY METHOD FOR THE ISOLATION OF THROMBIN-RESISTANT PEPTIDES WAS DEVELOPEDTHROMBIN-RESISTANT PEPTIDES WAS DEVELOPED

MAJORITY OF THE CLONES ARE EITHER FULLY MAJORITY OF THE CLONES ARE EITHER FULLY RESISTANT AGAINST THROMBIN OR COMPLETELY RESISTANT AGAINST THROMBIN OR COMPLETELY DIGESTED BY THROMBINDIGESTED BY THROMBIN

HIGH PERCENTAGE OF THROMBIN-RESISTANT HIGH PERCENTAGE OF THROMBIN-RESISTANT PEPTIDESPEPTIDES

HIGH PERCENTAGE OF FOLDED PEPTIDES IN A HIGH PERCENTAGE OF FOLDED PEPTIDES IN A RANDOM-PEPTIDE POPULATIONRANDOM-PEPTIDE POPULATION

OUTLOOK

• STRUCTURAL STUDIES OF MORE THROMBIN-RESISTANT PEPTIDES-cd measurements-nmr-crystals

• COMPUTATIONAL ANALYSIS OF THE PEPTIDE SEQUENCES-codon statistics-positional scanning-nearest-neighbor analysis-comparison with E. coli and Human small proteins

• ISOLATION OF FOLDED RNA FROM THE PHAGE LIBRARY•Insertion of T7 promoter in the current library•Selection with RNAse to select folded RNA