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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.