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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