Download Binding of Peptides and Peptide Analogs to DNA The Transcription

Bielefeld University
SFB 613
K2
Binding of Peptides and Peptide Analogs to DNA
The Transcription Factor PhoB from E. coli
N. Sewald1, D. Anselmetti2, R. Ros2
K. Wollschläger1, R. Eckel1, K. Gaus1, A. Körnig2, M. Schreiber1, D. Wesner2, M. McIntosh3, S. D. Wilking1, A. Becker3
1 Organische
und Bioorganische Chemie, Fakultät für Chemie
Experimentelle Biophysik, Fakultät für Physik
3 Genetik, Fakultät für Biologie
2
EMSA – Electrophoretic Mobility Shift Assay
Project Objectives
•
Protein
Studies of molecular recognition between DNA and transcription factors or transcription
factor epitopes on the single molecule level
•
DNA
Isolation of the wildtype and mutated DNA-binding domain (DBD) of E. coli PhoB using
intein mediated protein purification
•
•
•
Peptide synthesis using solid phase peptide synthesis (SPPS)
•
Structural investigations using NMR and CD (circular dichroism)
-
R203A
+
+
+
DNA : Protein
1:0
1:100
1:250
1:500
1:1000
DNA-Protein
complex
Unbound
DNA
Ligation reaction to introduce a cysteine residue for immobilisation in AFM-experiments
Determination of binding contribution of amino-acid residues that are supposed to be
involved in the binding to DNA
WT
DNA-Protein
complex
18 bp DNA labeled with Cy3, 10 % polyacrylamide/TBE gel,
visualized with Typhoon scanner
Unbound
DNA
FCS – Fluorescence Correlation Spectroscopy
The Transcription Factor PhoB
•
•
Part of the phosphate metabolism
Composed of two domains:
regulatory domain
Æ activation by phosphorylation
DNA binding domain
Æ helix-turn-helix motif binds to
specific DNA sequences
Synthesized peptide (190-209)
Æ α3 helix important for DNA binding
•
•
Confocal microscopy
Nanomolar concentration of
fluorescent ligand molecules
Single molecule detection
Autocorrelation analysis of
fluorescence intensity
fluctuations ⇒ G(t)
Chitin
HS
NH2
Protein
O
Intein
oB
of Ph
O
OH
S
Protein
MESNA
O
DBD of PhoB
Protein: SH-group of the ligated cysteine immobilized to
a gold surface
AFM tip
PEG linker
DNA
DBD of PhoB
koff
Peptide[1]
PhoB (190-209)
τ [s]
koff [s-1]
protein
τ [s]
wildtype
3,1 ± 2,1
0,32
0,0025 ± 0,0021
400
R193A
0,071 ± 0,053
14
0,012 ± 0,008
83
H198A
49,5 ± 21,2
0,020
0,760 ± 0,250
1
R203A
—
—
—
—
160
120
peptide:
wildtype
R193A
H198A
protein:
wildtype
R193A
H198A
140
120
Force / pN
Force / pN
220
230
240
250
260
λ [nm]
Peptides show α-helical structure in
Trifluorethanol (TFE); random
structure in buffer (pH 7,4, 10 mM
Na2HPO4, 5 mM NaCl)
4,0E+05
2,0E+05
0,0E+00
200
210
220
230
240
250
260
-2,0E+05
-4,0E+05
-6,0E+05
λ [nm]
270
280
290
300
DNA
DNA : wt 1:1
DNA : wt 1:1,5
DNA : wt 1:2
5,0E+03
190
200
210
220
230
240
250
60
40
λ [nm]
-1,5E+04
CD spectra indicate no structural
differences between mutant and
wildtype protein
6,0E+05
4,0E+05
2,0E+05
0,0E+00
200
210
220
230
240
250
-2,0E+05
-4,0E+05
-6,0E+05
λ [nm]
260
270
280
290
100
CD spectra indicate structural change in the DNA upon binding of the wt protein
•
•
•
•
Proteins bind in EMSA and AFM experiments, no structural differences are seen in CD spectra
•
•
•
•
Determination of on-rates in SPR and FCS experiments
Structure of the DNA changes upon protein binding
Peptides bind in AFM experiments, α-helical structure in TFE, no defined structure in buffer
Results of the AFM experiments with proteins and peptides are comparable
Competition AFM experiments with peptides
Investigation of amino acid residues important for specific DNA binding
Synthesis and analysis of truncated und cyclic proteins
80
60
0
1000
10000
Loading rate / pN s
[1]
[2]
[3]
[4]
Acknowledgement
20
0
100
100000
-1
300
DNA
DNA : R203A 1:1
DNA : R203A 1:1,5
DNA : R203A 1:2
40
20
10
260
-5,0E+03
Conclusions and Outlook
DNA: 360 bp Pho-box, SH-labeled
[s-1]
80
210
wt protein
R193A
H198A
R203A
Peptide: 1,8-diamino-3,6-dioxaoctane-linker connected
to cross-linker BS3(bis(sulfosuccinimidyl)suberate)
immobilized to an amino-functionalized mica surface
Gold surface
100
200
-2,0E+04
6,0E+05
Atomic Force Spectroscopy (AFM)
190
-1,0E+04
OH
NH
Ligation with
Cysteine
C
0,0E+00
180
SH
O
Ligation with
Cysteine
1,0E+04
-3,0E+04
NH2
Chitin
Elution
DBD of PhoB
OH
molar ellipticity [deg cm2 dmol-1]
-
Thiol-Induced
Cleavage with
MESNA
DBD
O-
2,0E+04
molar ellipticity [deg cm2 dmol-1]
Intein
O
O
S
molar ellipticity [deg cm2 dmol-1]
DBD of PhoB
O
S
O
molar ellipticity [deg cm2 dmol-1]
Protein
1,5E+04
wt peptide in TFE
wt peptide in buffer
3,0E+04
Intein
Load and Wash
D
CD – Circular Dichroism
Protein Modification by Native Ligation
DBD of PhoB
Average diffusion time through Diffusion time of Cy3-labeled
DNA increases with rising
the focus τ increases upon
concentrations of PhoBcomplex formation
protein
1000000
10
100
1000
10000
Loading rate / pN s-1
100000
1000000
S. Braun, H. Hansen and F. Hofmann for experimental assistance. This work was supported
by the DFG (SFB 613).
R. Eckel, S. D. Wilking, A. Becker, N. Sewald, R. Ros, D. Anselmetti “Single Molecule Experiments in Synthetic Biology – A New Approach for the Affinity Ranking of DNA-binding
Peptides” Angew. Chem. 117 (2005) 3989-3993; Angew. Chem. Int. Ed. Engl. 44 (2005) 3921-3924.
F. W. Bartels, M. McIntosh, A. Fuhrmann, C. Metzendorf, P. Plattner, N. Sewald, D. Anselmetti, R. Ros, A. Becker “Effector-stimulated single molecule protein-DNA interactions of a
quorum sensing system in Sinorhizobium meliloti” Biophys. J., 92 (2007) 4391-4400.
N. Sewald, S. D. Wilking, R. Eckel, S. Albu, K. Wollschläger, K. Gaus, A. Becker, F. W. Bartels, R. Ros, D. Anselmetti “Probing DNA – Peptide Interaction Forces at the Single Molecule
Level” J. Pept. Sci., 12 (2006) 836-842.
D. Anselmetti, F.W. Bartels, A. Becker, B. Decker, R. Eckel, M. McIntosh, J. Mattay, P. Plattner, R. Ros, C. Schäfer, N Sewald “Reverse Engineering of an Affinity-Switchable Molecular
Interaction Characterized by AFM Single Molecule Force Spectroscopy” submitted.