Lab Meeting:   “Turning Houseflies into Fireflies” Szostak Lab   Howard Hughes Medical Institute, and Department of Molecu...
Problem: <ul><li>How can one track the locations and relative concentrations of molecules (esp. those of prebiotic interes...
Background:  Biosensors   <ul><li>Indicate the presence of molecules </li></ul><ul><li>Specificity to targets & signal tra...
Background:   E. coli  bPBPs <ul><li>b acterial  P eriplasmic  B inding  P roteins </li></ul><ul><ul><li>2 domains linked ...
Background:   E. coli  bPBPs <ul><li>Changes in fluorescence intensity indicate ligand binding </li></ul><ul><ul><li>Envir...
ABC Transport System: <ul><li>A TP  B inding  C assette </li></ul><ul><ul><li>Periplasmic Protein-Dependent Transport Syst...
    
 
Labeling with Acrylodan <ul><li>6-acryloyl-2-dimethylaminonaphthalene  </li></ul><ul><li>Molecular Formula:  C 15 H 15 NO ...
Background:  Biosensors   <ul><li>Objective:  Synthesize a collection of binding proteins that exploits a common signal tr...
Background:  Biosensors   <ul><li>Objective:  Synthesize a collection of binding proteins that exploits a common signal tr...
Binding Proteins of Interest 34.470 kD 28.460 kD 34.473 kD   40.775 kD    26.141 kD 17.822 kD   31.000 kD 24.988 kD   29.7...
Binding Proteins of Interest 37.696 kD 28.474 kD 35.832 kD   40.928 kD    59.239 kD 33.334 kD   31.000 kD 24.988 kD   29.7...
phoS :  Cloning with pET100/TOPO <ul><li>Champion TM  pET Directional TOPO® Expression Kit (Invitrogen) </li></ul>Digest C...
PhoS:   Mass Spectrophotometry <ul><li>PhoS = 35.832 kD </li></ul><ul><li>His-Tag = 3 kD </li></ul>38,832 D  
hisJ :  Cloning with pET100/TOPO <ul><li>HisJ : V163C </li></ul>↓   Digest Confirmation Gel Sequenced & Expressed
HisJ Purification Process <ul><li>1L culture </li></ul><ul><li>Ni-NTA column </li></ul><ul><li>Dialysis:  50 mM HEPES, 50 ...
Affinity of HisJ:  Titrations <ul><li>20 mM MOPS, 100 mM NaCl pH 6.9 </li></ul><ul><li>20 mM NaH 2 PO 4 , 100 mM NaCl pH =...
sbp :  Cloning with TOPO/pET100 <ul><li>Sbp:  L65C </li></ul>Digest Confirmation Gel Sequenced & Expressed ↓
Sbp Purification Process <ul><li>1L culture </li></ul><ul><li>Ni-NTA column </li></ul><ul><li>Dialysis:  20 mM Tris-HCl, p...
rbp :  Cloning with TOPO/pET100 <ul><li>Rbp:  A234C </li></ul>Digest Confirmation Gel Sequenced & Expressed ↓
Rbp Purification Process <ul><li>Expressed </li></ul><ul><li>Ni-NTA column </li></ul><ul><li>M.W. = 28 kD </li></ul><ul><l...
And the Others? More of the same… Sbp Rbp HisJ MglB MalE
What can be eliminated: <ul><li>Buffer pH is not near pI </li></ul><ul><li>Storage (never frozen, tested immediately & sti...
SDS-PAGE
Still More Proteins… DppA:  Dipeptide Binding Protein (D450C) Digest Confirmation Gel Sequenced & Expressed ↓
DppA:  D450C Mutation <ul><li>Very difficult to sequence mutagenized segment (N’s); 3 different sequencing primers! </li><...
Still More Proteins… MalE:  Maltose Binding Protein (D95C) Digest Confirmation Gel Sequenced & Expressed ↓   ↓
Still More Proteins… MglB:  Glucose-Galactose Binding Protein (H152C) Digest Confirmation Gel Sequenced & Expressed ↓   ↓
Still More Proteins… YbeJ (GltI):   Glutamate-Aspartate Binding Protein (H152C) Digest Confirmation Gel Sequenced & Expres...
Still More Proteins… GlnH:  Glutamine Binding Protein (Y163C) FhuD:  Fe 3+  Binding Protein
Future Plans <ul><li>High Priority: </li></ul><ul><ul><li>Sbp, Rbp, MglB, MalE & HisJ:  Repair defective binding measureme...
Acknowledgments <ul><li>Jack </li></ul><ul><li>Sheref  </li></ul><ul><li>Mark </li></ul><ul><li>Raphael </li></ul><ul><li>...
??Questions??
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Bacterial Periplasmic Binding Proteins as Biosensors in Liposomes

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Description of project to engineer fluorophore bacterial periplasmic binding proteins for use as biosensors in liposomes

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Bacterial Periplasmic Binding Proteins as Biosensors in Liposomes

  1. 1. Lab Meeting: “Turning Houseflies into Fireflies” Szostak Lab Howard Hughes Medical Institute, and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114
  2. 2. Problem: <ul><li>How can one track the locations and relative concentrations of molecules (esp. those of prebiotic interest) in liposomes? </li></ul>http://bioweb.wku.edu/courses/biol22000/2Bonds/ http://bioweb.wku.edu/pix/Pix.htm
  3. 3. Background: Biosensors <ul><li>Indicate the presence of molecules </li></ul><ul><li>Specificity to targets & signal transduction: ligand binding  detectable physical changes </li></ul><ul><li>Should be reagentless: do not change composition as a consequence of making the measurement </li></ul><ul><li>Ligand-Specific Periplasmic Binding Proteins </li></ul><ul><ul><li>Superfamily of macromolecules that facilitate the active transport of ions, sugars and amino acids </li></ul></ul><ul><ul><li>Can be engineered to sense these substrates </li></ul></ul>
  4. 4. Background: E. coli bPBPs <ul><li>b acterial P eriplasmic B inding P roteins </li></ul><ul><ul><li>2 domains linked by a hinge region </li></ul></ul><ul><ul><li>Ligand-binding site is located at the interface between them. </li></ul></ul><ul><li>2 conformations that interconvert via hinge bending </li></ul><ul><ul><li>Ligand-free open </li></ul></ul><ul><ul><li>Liganded closed </li></ul></ul><ul><li>Conformational change attributed to reporter functions </li></ul><ul><li>Fluorophore (Acrylodan) conjugates of bPBPs specific for various ligands </li></ul>Felder, C.B., et al. (1999) AAPS PharmSci. 1 (2). 1-20. www.botgard.ucla.edu /. ../b0567tx.html
  5. 5. Background: E. coli bPBPs <ul><li>Changes in fluorescence intensity indicate ligand binding </li></ul><ul><ul><li>Environmentally-sensitive fluorophores are placed in locations that undergo conformational change (local  global) </li></ul></ul><ul><ul><li>Cys mutations are placed at these sites so that changes in fluorescence may be directly linked to ligand binding. </li></ul></ul><ul><li>Sites identified by De Lorimier et al. </li></ul><ul><ul><li>Protein Science (2002), 11:2655–2675. </li></ul></ul><ul><ul><li>Closed structure examination, open vs. closed & structural/sequence homology </li></ul></ul>Hinge Mutated Residue Bound Ligand De Lorimier et al. Protein Science. 11:2655–2675.
  6. 6. ABC Transport System: <ul><li>A TP B inding C assette </li></ul><ul><ul><li>Periplasmic Protein-Dependent Transport System (G-) </li></ul></ul><ul><ul><li>Binding Lipoprotein-Dependent Transport System (G+) </li></ul></ul><ul><li>bPBPs are initial receptors in active transport across cellular membranes &/or chemotaxis </li></ul><ul><li>Each binds a specific substrate (e.g. sugar, amino acid, or ion) with high affinity. </li></ul><ul><li>3 Types of Constituents: Genes typically found in an operon </li></ul><ul><ul><li>2 integral membrane proteins (permeases) each having 6- transmembrane segment </li></ul></ul><ul><ul><li>2 peripheral membrane proteins that bind & hydrolyze ATP (most evolutionarily conserved) </li></ul></ul><ul><ul><li>A periplasmic (or lipoprotein) substrate-binding protein (the part we seek to exploit in our assays) </li></ul></ul>
  7. 7.     
  8. 8.  
  9. 9. Labeling with Acrylodan <ul><li>6-acryloyl-2-dimethylaminonaphthalene </li></ul><ul><li>Molecular Formula: C 15 H 15 NO </li></ul><ul><li>Molecular Weight: 225.29 </li></ul><ul><li>Labeling Rxn: </li></ul><ul><ul><li>36.4 μM protein stock (0.014 g/ml) for 300 μL protein </li></ul></ul><ul><ul><li>Add 2.16 μl of 50 mM TCEP (50 mM TCEP; 10 mM dye) </li></ul></ul><ul><ul><li>Add 10.8 μl of 10 mM dye (0.001g/0.5ml) </li></ul></ul><ul><ul><li>Leave mixture in fridge overnight. </li></ul></ul>Emission: 500 nm Excitation: 390 nm
  10. 10. Background: Biosensors <ul><li>Objective: Synthesize a collection of binding proteins that exploits a common signal transduction mechanism </li></ul><ul><ul><li>Fluorophore reports binding of specific molecular targets in liposomes </li></ul></ul><ul><ul><li>Amino acids, cations, anions, dipeptides & sugars </li></ul></ul>
  11. 11. Background: Biosensors <ul><li>Objective: Synthesize a collection of binding proteins that exploits a common signal transduction mechanism </li></ul><ul><ul><li>Fluorophore reports binding of specific molecular targets in liposomes </li></ul></ul><ul><ul><li>Amino acids, cations, anions, dipeptides & sugars </li></ul></ul>http://www.darnellworks.com/a52/nr0007.htm
  12. 12. Binding Proteins of Interest 34.470 kD 28.460 kD 34.473 kD 40.775 kD   26.141 kD 17.822 kD 31.000 kD 24.988 kD 29.714 kD  57.536 kD 33.196 kD Protein Size L65C sbp Sulfate A234C rbp Ribose phoS malE hisJ mglB gltI/ybeJ glnH fhuD dppA araF Gene F126C Glutamate-Aspartate S164C (S197C) Phosphate D95C Maltose V163C Histidine H152C Glucose-Galactose Y163C Glutamine Iron D450C Dipeptide K301C, C64A Arabinose Mutations Binding Protein
  13. 13. Binding Proteins of Interest 37.696 kD 28.474 kD 35.832 kD 40.928 kD   59.239 kD 33.334 kD 31.000 kD 24.988 kD 29.714 kD  66.8982 kD 33.196 kD Protein Size L65C sbp Sulfate Functional A234C rbp Ribose Functional phoS malE hisJ mglB gltI/ybeJ glnH fhuD dppA araF Gene Functional Functional Functional Functional Expressed Cloned WT Cloned WT Purified 1 st of double mutant Status F126C Glutamate-Aspartate S164C (A197C) Phosphate D95C Maltose V163C Histidine H152C Glucose-Galactose Y163C Glutamine E203C Iron D450C Dipeptide K301C, C64A Arabinose Mutations Binding Protein
  14. 14. phoS : Cloning with pET100/TOPO <ul><li>Champion TM pET Directional TOPO® Expression Kit (Invitrogen) </li></ul>Digest Confirmation Gel Sequenced & Expressed ↓ PhoS: S164C (A197C)
  15. 15. PhoS: Mass Spectrophotometry <ul><li>PhoS = 35.832 kD </li></ul><ul><li>His-Tag = 3 kD </li></ul>38,832 D 
  16. 16. hisJ : Cloning with pET100/TOPO <ul><li>HisJ : V163C </li></ul>↓ Digest Confirmation Gel Sequenced & Expressed
  17. 17. HisJ Purification Process <ul><li>1L culture </li></ul><ul><li>Ni-NTA column </li></ul><ul><li>Dialysis: 50 mM HEPES, 50 mM NaCl, pH = 7.4 </li></ul><ul><li>M.W. = 26 kD </li></ul><ul><li>Labeled with Acrylodan </li></ul>50 kD
  18. 18. Affinity of HisJ: Titrations <ul><li>20 mM MOPS, 100 mM NaCl pH 6.9 </li></ul><ul><li>20 mM NaH 2 PO 4 , 100 mM NaCl pH = 6.9 </li></ul>
  19. 19. sbp : Cloning with TOPO/pET100 <ul><li>Sbp: L65C </li></ul>Digest Confirmation Gel Sequenced & Expressed ↓
  20. 20. Sbp Purification Process <ul><li>1L culture </li></ul><ul><li>Ni-NTA column </li></ul><ul><li>Dialysis: 20 mM Tris-HCl, pH = 8.0, cholestryramine (Dowex 1X2-100) resin </li></ul><ul><li>M.W. = 35 kD </li></ul><ul><li>Labeled with Acrylodan </li></ul>
  21. 21. rbp : Cloning with TOPO/pET100 <ul><li>Rbp: A234C </li></ul>Digest Confirmation Gel Sequenced & Expressed ↓
  22. 22. Rbp Purification Process <ul><li>Expressed </li></ul><ul><li>Ni-NTA column </li></ul><ul><li>M.W. = 28 kD </li></ul><ul><li>Found in pellet? </li></ul><ul><ul><li>Run through column with urea </li></ul></ul><ul><ul><li>May need refolding… </li></ul></ul>
  23. 23. And the Others? More of the same… Sbp Rbp HisJ MglB MalE
  24. 24. What can be eliminated: <ul><li>Buffer pH is not near pI </li></ul><ul><li>Storage (never frozen, tested immediately & still dysfunctional; also tried 5 & 30% glycerol when frozen) </li></ul><ul><li>Sequences (Mutation clearly sequenced & no other errors) </li></ul><ul><li>Mass (Mass Spec/SDS-PAGE) </li></ul><ul><li>Column contamination (each protein has it’s own Ni-NTA column) </li></ul>
  25. 25. SDS-PAGE
  26. 26. Still More Proteins… DppA: Dipeptide Binding Protein (D450C) Digest Confirmation Gel Sequenced & Expressed ↓
  27. 27. DppA: D450C Mutation <ul><li>Very difficult to sequence mutagenized segment (N’s); 3 different sequencing primers! </li></ul><ul><li>Expressed to test functionality, SDS-PAGE & M.S. </li></ul>
  28. 28. Still More Proteins… MalE: Maltose Binding Protein (D95C) Digest Confirmation Gel Sequenced & Expressed ↓ ↓
  29. 29. Still More Proteins… MglB: Glucose-Galactose Binding Protein (H152C) Digest Confirmation Gel Sequenced & Expressed ↓ ↓
  30. 30. Still More Proteins… YbeJ (GltI): Glutamate-Aspartate Binding Protein (H152C) Digest Confirmation Gel Sequenced & Expressed ↓
  31. 31. Still More Proteins… GlnH: Glutamine Binding Protein (Y163C) FhuD: Fe 3+ Binding Protein
  32. 32. Future Plans <ul><li>High Priority: </li></ul><ul><ul><li>Sbp, Rbp, MglB, MalE & HisJ: Repair defective binding measurement </li></ul></ul><ul><ul><ul><li>Investigate effect of salts, pH (unlikely) </li></ul></ul></ul><ul><ul><ul><li>Possible contamination? </li></ul></ul></ul><ul><ul><li>DppA: Test kinetics (M.S.) </li></ul></ul><ul><ul><li>PhoS: Keep working or buy? </li></ul></ul><ul><li>Lower Priority: </li></ul><ul><ul><li>AraF: C64A (K301C done) </li></ul></ul><ul><ul><li>YbeJ: F126C </li></ul></ul><ul><ul><li>GlnH: Y163C </li></ul></ul><ul><ul><li>FhuD: E203C </li></ul></ul><ul><li>Devise a new assay system? </li></ul><ul><li>Concerns: </li></ul><ul><li>- Cost to produce “in-house” </li></ul><ul><li>Stability </li></ul><ul><li>Ease of use/time to produce </li></ul><ul><li>System limitations </li></ul>
  33. 33. Acknowledgments <ul><li>Jack </li></ul><ul><li>Sheref </li></ul><ul><li>Mark </li></ul><ul><li>Raphael </li></ul><ul><li>Yollette </li></ul><ul><li>Florian </li></ul>
  34. 34. ??Questions??
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